HK1167875A

HK1167875A - Novel cell lines and methods

Info

Publication number: HK1167875A
Application number: HK12108675.1A
Authority: HK
Inventors: K．夏柯达; D．梭茱克; P．M．沙阿
Original assignee: 卓莫赛尔公司
Priority date: 2009-02-02
Filing date: 2010-02-01
Publication date: 2012-12-14

Abstract

The invention relates to novel cells and cell lines, and methods for making and using them.

Description

Novel cell lines and methods

The present application claims the benefit of U.S. provisional application No.61/149,321 filed on 2/2009, U.S. provisional application No. 361/149,318 filed on 2/2009, U.S. provisional application No.61/149,324 filed on 2/2009, U.S. provisional application No.61/149,311 filed on 2/2009, U.S. provisional application No.61/235,181 filed on 8/19/2009, and U.S. provisional application No.61/230,536 filed on 7/31/2009, each of which is incorporated herein by reference in its entirety.

Sequence listing

This application includes the sequence listing that has been filed by EFS-Web and is incorporated by reference herein in its entirety. The ASCII copy created on 1/2/2010 was named 0022980025 seqlist.

Technical Field

The present invention relates to novel cells and cell lines and methods for making and using them. In particular embodiments, the invention relates to cells and cell lines that stably express complex targets (complextargets). The invention also provides methods of making such cells and cell lines. The cells and cell lines provided herein are useful for identifying modulators of such complex targets.

Background

Currently, the average failure rate in the industry for pharmaceutical companies' drug development programs is reported to be about 98%. While this includes failures at all stages of the process, the high failure rate means that any improvement in process efficiency is urgently needed.

One factor contributing to the high failure rate is the lack of cell lines expressing therapeutic targets used in cell-based functional assays during drug development. Undoubtedly, studies using cell-based assays, particularly drug development studies, would benefit from the cells and cell lines used in cell-based assays.

Therefore, there is an urgent need to quickly and efficiently establish cell-based assays for more rapid development of new and improved drugs. Preferably, for more efficient drug development, the assay system should provide a predictor of the effect of more physiologically relevant modulators in vivo.

In addition to the need for cell-based assays, there is a need for improved cells for protein production, cell-based therapies, and various other uses.

Thus, there is an urgent need for cells and cell lines that express functional proteins or RNAs of interest.

Taste Receptor Cells (TRCs) can be found in several specialized regions in the mouth, including the tongue, parts of the palate, epiglottis, larynx and pharynx. On the tongue, TRCs are organized into a population of cells called taste buds. The taste bud consists of a single apical pore in which the microvilli of the TRC are in contact with a tastant (tastant) present in the oral cavity. On the tongue, taste buds are buried in three types of specialized epidermal structures. The fungiform papillae are distributed over the first two thirds of the tongue. The foliated papillae, which develops well at birth but regresses with age, is found on the side of the posterior third of the tongue. Between 7 and 9 circumvallate papillae are located on the posterior tongue as far as the posterior portion near the sulcus. In addition to organized 'classic' TRCs in taste buds, clusters of chemosensory cells (chemosensory cells) or individual chemosensory cells (somatual chemosensory cells) are found in the non-lingual epithelium of the lung and intestine.

Sweet taste receptor

The perception of sweetness is mediated by a heteromeric G protein-coupled receptor (GPCR) consisting of two subunits, TASR2(T1R2) and TASR3(T1R 3). This receptor is called sweet taste receptor. Two subunits of receptors are members of the class C GPCR subfamily and have a large N-terminal extracellular domain, commonly referred to as the venus flytrap domain. The T1R subunit can be coupled to G proteins, alpha transducins (transducins) or alpha gustducins (gustducins), through which they activate the phospholipase C (PLC) beta 2-dependent pathway to increase intracellular Ca²⁺And (4) concentration. They may also activate cAMP-dependent pathways.

Sweet taste receptors detect a variety of sweet chemicals, including single carbohydrates (e.g., sugars), amino acids, peptides, proteins, and synthetic sweeteners. Sweet taste receptors are sensitive to both natural and artificial sweeteners. In view of the chemical structures of a variety of known activation receptors, it has been proposed that there be multiple binding sites in the receptor, including sites in the transmembrane region and sites on T1R3, which T1R3 serves as a subunit common to umami receptors.

Sweet taste receptors are also implicated in disorders such as obesity and diabetes, as such receptors appear to play an important role in nutrient detection and sensation. Taste receptors are expressed in nutrient detection regions of the human proximal small intestine, where evidence suggests that they play a role in detecting nutrients in the intestinal lumen. In this region, there is a highly synergistic expression of sweet taste receptors and gustducin (a G protein involved in intracellular taste signaling), more specifically in endocrine cells of the intestine. The function of such sweet taste receptors may thus show ligand-mediated control similar to other G protein-coupled receptors, i.e. they may lose their activity and/or expression in the presence of their high concentration of ligand. This can produce intestinal 'taste' signaling in response to dynamic metabolic changes in glucose concentration in the blood and lumen. Thus, sweet taste receptors and their regulation in the gut may have important roles in diet, appetite, and in the treatment of a variety of diseases such as obesity and diabetes.

Activation of the intestinal sweet receptor by natural sugars and artificial sweeteners also results in increased expression of apical glucose transporter (GLUT 2) and other glucose transporters. For example, artificial sweeteners are nutritionally active, as they may signal a functional taste acceptance system (functional taste acceptance system) to increase sugar absorption during meals, which may be a significant finding in nutrition and appetite and thus in the potential treatment of malnutrition and eating disorders. The ever-increasing levels of top GLUT2 resulted in increased sugar absorption and was characteristic of experimental diabetes and the state of insulin resistance induced by fructose and fat. In addition, activation of sweet taste receptors in neuroendocrine cells results in the release of glucagon-like peptide (GLP-1) and possibly other digestive regulators. Overall, sweet taste receptors in the intestine play an important role in the perception of the nutritional value of the intestinal lumen contents and help coordinate the body's responses through regulated absorption and digestion. These findings suggest that sweet taste receptors may be useful as potential targets for modulators for the treatment of obesity and diabetes.

Umami taste receptor

The umami (umami) sensation is mediated by a heteromeric GPCR consisting of 2 subunits TASR1(T1R1) and TASR3(T1R 3). This receptor is called umami receptor. Both subunits of the receptor are members of the class C GPCR subfamily and have a large N-terminal extracellular domain commonly referred to as the venus flytrap domain. The T1R subunit can be coupled to G protein alpha transducin or alpha gustducin, through which they can activate the phospholipase C (PLC) 2-dependent pathway to increase intracellular Ca²⁺And (4) concentration. They may also activate the cAMP-dependent pathway. Such receptors detect a wide variety of palatable chemicals including L-amino acids and monosodium glutamate (MSG). T1R1 has also been shown to bind to disodium 5' -inosinate (I)MP) and other nucleotides, known umami enhancers.

Umami receptors are also expressed in the nutrient detection region of the human proximal small intestine where they are thought to play a role in detecting nutrients in the intestinal lumen. In this region, particularly in neuroendocrine cells, there is a highly synergistic expression of umami receptors and taste transduction proteins (G proteins involved in intracellular taste signaling). Activation of the gut umami receptor by amino acids results in modulation of the apical oligopeptide transporter PepT 1. In general, umami receptors in the intestine play an important role in sensing the nutritional value of the intestinal lumen contents and help coordinate the body's responses through regulated absorption and digestion. These findings suggest that umami receptors may be useful as potential targets for modulators for the treatment of obesity and diabetes.

Bitter taste receptors

Bitter taste receptors are G protein-coupled receptors (GPCRs) expressed on the surface of taste receptor cells and coupled to second messenger pathways. The TAS2R receptor can, for example, be coupled to transducin (e.g., GNAT1, GNAT2, and guanine nucleotide binding protein g (t)) or gustducin (e.g., GNAT3 guanine nucleotide binding protein and alpha transducin 3) through which they can activate phosphodiesterase and phospholipase c (plc) beta 2-dependent pathways to increase intracellular Ca²⁺And (4) concentration. The TAS2R receptor can also be coupled to human GNA15 (guanine nucleotide binding protein (G protein) α 15(Gq species; synonyms GNA16) and mouse G α 15 and their chimeric proteins G α 15-GNA15 (also known as G α 15-Ga 16).

Human bitter taste is mediated by about 25 members of the human TAS2 receptor (hTAS2R) gene family. Bitter taste receptors are also important in a range of physiological contexts, in addition to their role in taste. For example, taste receptor agonists induce secretory responses in enteroendocrine cells in vitro as well as in animals, and induce neuronal activation. Thus, all bitter taste receptor family members are important clinical targets for managing a variety of conditions associated with the detection of bitter tastants.

The discovery of new and improved compounds that specifically target taste receptors (e.g., sweet, umami, and bitter taste receptors) and thereby modulate their activity has been hampered by the lack of robust physiologically relevant cell-based systems, more particularly such systems that are amenable to high throughput format processing to identify and test taste receptor modulators (e.g., sweet, umami, and bitter taste receptor modulators). Such cell-based systems are preferred for drug discovery and validation because they provide a functional assay for the compound relative to cell-free systems that provide only binding assays. Furthermore, cell-based systems have the advantage of simultaneously testing cytotoxicity. Ideally, the cell-based system should also stably and constitutively express the target protein. It is also desirable that cell-based systems be reproducible. The present invention addresses these problems in various embodiments in the context of providing cells and cell lines that stably express taste receptors, e.g., sweet, bitter, or umami receptors, in physiologically relevant forms and in methods of using such cells and cell lines to identify modulators of taste receptors, e.g., sweet, bitter, or umami receptors.

Disclosure of Invention

In certain embodiments, the invention provides a cell that expresses a heterodimeric protein of interest from an introduced nucleic acid encoding at least one subunit of a heterodimeric protein of interest, the cell being characterized in that it produces the heterodimeric protein of interest in a form suitable for use in a functional assay, wherein the protein of interest does not comprise a protein tag, or the protein is produced consistently and reproducibly in a form such that the cell has a Z' factor of at least 0.4 in the functional assay, or the cell is cultured in the absence of selective pressure, or any combination thereof.

In certain embodiments, the invention provides a cell that expresses a heterodimeric protein of interest, wherein the cell is engineered to activate transcription of an endogenous nucleic acid encoding at least one subunit of the heterodimeric protein of interest, the cell being characterized in that it produces the heterodimeric protein of interest in a form suitable for use in a functional assay, wherein the protein of interest does not comprise a protein tag, or the protein is produced consistently and reproducibly in a form such that the cell has a Z' factor of at least 0.4 in the functional assay, or the cell is cultured in the absence of selective pressure, or any combination thereof.

In certain embodiments, the invention provides a cell that expresses a heterodimeric protein of interest from an introduced nucleic acid encoding at least one subunit of a heterodimeric protein of interest, the cell being characterized in that it produces the protein of interest in a form that is or is capable of becoming biologically active, wherein the cell is cultured in the absence of selective pressure.

In certain embodiments, the invention provides a cell that expresses a heterodimeric protein of interest, wherein the cell is engineered to activate transcription of an endogenous nucleic acid encoding at least one subunit of the heterodimeric protein of interest, the cell being characterized in that it produces the protein of interest in a form that is or is capable of becoming biologically active, wherein the cell is cultured in the absence of selective pressure.

In certain embodiments, the nucleic acid encoding the second subunit of the heterodimeric protein of interest is endogenous. In other embodiments, a nucleic acid encoding a second subunit of the heterodimeric protein of interest is introduced. In still other embodiments, the protein of interest does not comprise a protein tag.

In certain embodiments, the heterodimeric protein of interest is selected from the group consisting of: ion channels, G protein-coupled receptors (GPCRs), tyrosine receptor kinases, cytokine receptors, nuclear steroid hormone receptors, antibodies, biologicals, and immunoreceptors. In certain embodiments, the heterodimeric protein of interest is an antibody or a biologic. In certain embodiments, the heterodimeric protein of interest is selected from the group consisting of: sweet taste receptors and umami taste receptors. In certain embodiments, the heterodimeric protein of interest does not have a known ligand. In other embodiments, there are no known assays to detect functional expression of the heterodimeric protein of interest.

In certain embodiments, the heterodimeric protein of interest is not expressed in the same type of cell. In certain embodiments, the cell is a mammalian cell.

In certain embodiments, the cell is further characterized in that it has additional desired properties selected from the group consisting of: signal to noise ratio greater than 1, stable over time, growth without selective pressure without loss of expression, physiological EC₅₀Value and physiological IC₅₀The value is obtained. In certain embodiments, the heterodimeric protein of interest is produced consistently and reproducibly in a form and for a period of time selected from: at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, and at least 9 months. In certain embodiments, the functional assay is selected from: cell-based assays, fluorescent cell-based assays, high throughput screening assays, reporter cell-based assays, G protein-mediated cell-based assays, and calcium flux cell-based assays. In certain embodiments, the functional assay is a membrane potential assay, ELISA, mass spectrometry, biochemical characterization of a protein of interest, cell growth assay, viability assay, cell specification assay, or the ability of protein production. In other embodiments, the cells are suitable for use in cell-based high-throughput screening.

In certain embodiments, the selection pressure is an antibiotic. In other embodiments, the cells express the heterodimeric protein in the absence of selective pressure for at least 15 days, 30 days, 45 days, 60 days, 75 days, 100 days, 120 days, or 150 days.

In certain embodiments, the invention provides a cell that expresses a heterodimeric protein of interest, wherein the heterodimeric protein of interest comprises at least 3 subunits, wherein at least one subunit of the heterodimeric protein of interest is encoded by an introduced nucleic acid, said cell being characterized in that it produces the heterodimeric protein of interest in a form suitable for use in a functional assay, wherein the protein of interest does not comprise a protein tag, or the protein is produced consistently and reproducibly in a form such that the cell has a Z' factor of at least 0.4 in the functional assay, or the cell is cultured in the absence of selective pressure, or any combination thereof.

In certain embodiments, the invention provides a cell that expresses a heterodimeric protein of interest, wherein the heterodimeric protein of interest comprises at least 3 subunits, wherein the cell is engineered to activate transcription of an endogenous nucleic acid encoding at least one subunit of the heterodimeric protein of interest, the cell being characterized in that it produces the heterodimeric protein of interest in a form suitable for use in a functional assay, wherein the protein of interest does not comprise a protein tag, or the protein is produced consistently and reproducibly in a form such that the cell has a Z' factor of at least 0.4 in the functional assay, or the cell is cultured in the absence of selective pressure, or any combination thereof.

In certain embodiments, the invention provides a cell expressing a heterodimeric protein of interest, wherein the heterodimeric protein of interest comprises at least 3 subunits, wherein at least one subunit of the heterodimeric protein of interest is encoded by an introduced nucleic acid, said cell being characterized in that it produces the protein of interest in a form that is or is capable of becoming biologically active.

In certain embodiments, the invention provides a cell that expresses a heteromultimeric protein of interest wherein the heteromultimeric protein comprises at least 3 subunits, wherein the cell is engineered to activate transcription of an endogenous nucleic acid encoding at least one subunit of the heteromultimeric protein of interest, said cell being characterized in that it produces the protein of interest in a form that is or is capable of becoming biologically active.

In certain embodiments, the nucleic acid encoding at least one subunit of the heteromultimeric protein of interest is endogenous.

In certain embodiments, a nucleic acid encoding at least one subunit of a heterodimeric protein of interest is introduced.

In certain embodiments, the protein of interest does not comprise a protein tag.

In certain embodiments, the heteromultimeric protein of interest is selected from the group consisting of: ion channels, G protein-coupled receptors (GPCRs), tyrosine receptor kinases, cytokine receptors, nuclear steroid hormone receptors, and immunoreceptors. In certain embodiments, the heteromultimeric protein of interest is an antibody or a biologic. In other embodiments, the heteromultimeric protein of interest is selected from the group consisting of: GABA, ENaC and NaV. In certain embodiments, the heteromultimeric protein of interest does not have a known ligand. In other embodiments, there is no known assay to detect functional expression of the heteromultimeric protein of interest.

In certain embodiments, the heteromultimeric protein of interest is not expressed in the same type of cell. In other embodiments, the cell is a mammalian cell.

In certain embodiments, the cell is further characterized in that it has additional desirable properties selected from the group consisting of: a signal to noise ratio greater than 1, stable over time, growth without selective pressure without loss of expression, physiological EC50 values and physiological IC50 values. In other embodiments, the heterodimeric protein of interest is produced consistently and chargelly in a form for a period of time selected from: at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, and at least 9 months.

In certain embodiments, the functional assay is selected from the group consisting of: cell-based assays, fluorescent cell-based assays, high throughput screening assays, reporter cell-based assays, G protein-mediated cell-based assays, and calcium flux cell-based assays. In certain embodiments, the functional assay is a membrane potential assay, ELISA, mass spectrometry, biochemical characterization of the protein of interest, cell growth assay, viability assay, cell-specific assay, or the ability of protein production. In other embodiments, cells expressing the heteromultimeric proteins are suitable for use in cell-based high throughput screening.

In certain embodiments, the cell expressing the heteromultimeric protein is cultured in the absence of selective pressure. In certain embodiments, the selection pressure is an antibiotic. In other embodiments, the cell of claim 35 or 36, wherein the cell expresses the heteromultimeric protein in the absence of selective pressure for at least 15 days, 30 days, 45 days, 60 days, 75 days, 100 days, 120 days, or 150 days.

In certain embodiments, the invention provides a cell that expresses two or more proteins of interest from an introduced nucleic acid encoding at least one protein of interest, the cell being characterized in that it produces the protein of interest in a form suitable for use in a functional assay, wherein the protein of interest does not comprise a protein tag, or the protein is produced consistently and reproducibly in a form such that the cell has a Z' factor of at least 0.4 in the functional assay, or the cell is cultured in the absence of selective pressure, or any combination thereof.

In certain embodiments, the invention provides a cell that expresses two or more proteins of interest, wherein the cell is engineered to activate transcription of an endogenous nucleic acid encoding at least one protein of interest, the cell being characterized in that it produces the heterodimeric protein of interest in a form suitable for use in a functional assay, wherein the protein of interest does not comprise a protein tag, or the protein is produced consistently and reproducibly in such a form that the cell has a Z' factor of at least 0.4 in the functional assay, or the cell is cultured in the absence of selective pressure, or any combination thereof.

In certain embodiments, the invention provides a cell that expresses two or more proteins of interest from an introduced nucleic acid encoding at least one protein of interest, the cell being characterized in that it produces the protein of interest in a form that is or is capable of becoming biologically active.

In certain embodiments, the invention provides a cell that expresses two or more proteins of interest, wherein the cell is engineered to activate transcription of an endogenous nucleic acid encoding at least one protein of interest, the cell being characterized in that it produces the protein of interest in a form that is or is capable of becoming biologically active.

In certain embodiments, at least one of the two or more proteins of interest is a dimeric protein. In other embodiments, the dimeric protein of interest is a homodimeric protein. In other embodiments, the dimeric protein of interest is a heterodimeric protein. In certain embodiments, at least one of the two or more proteins of interest is a multimeric protein. In other embodiments, the multimeric protein of interest is a homomultimeric protein. In other embodiments, the multimeric protein of interest is a heteromultimeric protein.

In certain embodiments, one of the two or more proteins of interest is encoded by an endogenous nucleic acid. In other embodiments, one of the two or more proteins of interest is encoded by the introduced nucleic acid. In other embodiments, the protein of interest does not comprise a protein tag.

In certain embodiments, one of the two or more proteins of interest is selected from the group consisting of: ion channels, G protein-coupled receptors (GPCRs), tyrosine receptor kinases, cytokine receptors, nuclear steroid hormone receptors, antibodies, biologicals, and immunoreceptors. In certain embodiments, the two or more proteins of interest are independently antibodies or biologicals. In other embodiments, one of the proteins of interest does not have a known ligand. In other embodiments, there are no known assays to detect functional expression of two or more proteins of interest.

In certain embodiments, one of the two or more proteins of interest is not expressed in the same type of cell. In certain embodiments, the cell expressing two or more proteins is a mammalian cell.

In certain embodiments, the cell expressing the two or more proteins is further characterized in that it has other desired properties selected from the group consisting of: a signal to noise ratio greater than 1, stable over time, growth without selective pressure without loss of expression, physiological EC50 values, and physiological IC50 values.

In certain embodiments, two or more proteins of interest are produced consistently and reproducibly in a form, for a period of time selected from the group consisting of: at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, and at least 9 months.

In certain embodiments, the functional assay is selected from the group consisting of: cell-based assays, fluorescent cell-based assays, high throughput screening assays, reporter cell-based assays, G protein-mediated cell-based assays, and calcium flux cell-based assays. In certain embodiments, the functional assay is a membrane potential assay, ELISA, mass spectrometry, biochemical characterization of the protein of interest, cell growth assay, viability assay, cell-specific assay, or the ability of protein production. In other embodiments, cells expressing two or more proteins are suitable for use in cell-based high-throughput screening.

In certain embodiments, cells expressing two or more proteins are cultured in the absence of selective pressure. In certain embodiments, the selection pressure is an antibiotic. In certain embodiments, the cells express the two or more proteins in the absence of selective pressure for at least 15 days, 30 days, 45 days, 60 days, 75 days, 100 days, 120 days, or 150 days.

In certain embodiments, the invention provides a cell expressing at least one RNA of interest, wherein the RNA of interest is encoded by an introduced nucleic acid, the cell being characterized in that it produces the at least one RNA of interest in a form suitable for use in a functional assay, wherein the RNA of interest does not comprise a tag, or the RNA is produced consistently and reproducibly in a form such that the cell has a Z' factor of at least 0.4 in the functional assay, or the cell is cultured in the absence of selective pressure, or any combination thereof.

In certain embodiments, the invention provides a cell that expresses at least one RNA of interest, wherein the cell is engineered to activate transcription of an endogenous nucleic acid encoding the at least one RNA of interest, the cell being characterized in that it produces the at least one RNA of interest in a form suitable for use in a functional assay, wherein the RNA of interest does not comprise a tag, or the RNA is produced consistently and reproducibly in a form such that the cell has a Z' factor of at least 0.4 in the functional assay, or the cell is cultured in the absence of selective pressure, or any combination thereof.

In certain embodiments, the cell expresses at least two RNAs of interest. In other embodiments, the cell expresses at least three RNAs of interest. In certain embodiments, the cell also expresses an RNA encoded by the introduced nucleic acid. In certain embodiments, the RNA of interest is selected from: RNA encoding ion channels, RNA encoding G protein-coupled receptors (GPCRs), RNA encoding tyrosine receptor kinases, RNA encoding cytokine receptors, RNA encoding nuclear steroid hormone receptors, and RNA encoding immunoreceptors. In other embodiments, the RNA of interest is an RNA encoding an antibody or an RNA encoding a biological product.

In certain embodiments, the RNA of interest is not expressed in the same type of cell. In certain embodiments, the cell expressing the RNA of interest is a mammalian cell.

In certain embodiments, the cell expressing the RNA of interest is further characterized in that it has additional desired properties selected from the group consisting of: signal to noise ratio greater than 1, stable over time, growth without selective pressure without loss of expression, physiological EC50 values and physiological IC50 values. In other embodiments, the RNA of interest is produced consistently and reproducibly in a format, for a period of time selected from the group consisting of: at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, and at least 9 months.

In certain embodiments, the functional assay is selected from the group consisting of: cell-based assays, fluorescent cell-based assays, high throughput screening assays, reporter cell-based assays, G protein-mediated cell-based assays, and calcium flux cell-based assays. In other embodiments, the functional assay is a membrane potential assay, ELISA, mass spectrometry, biochemical characterization of the protein of interest, cell growth assay, viability assay, cell-specific assay, or the ability of protein production.

In certain embodiments, cells expressing an RNA of interest are suitable for cell-based high-throughput screening.

In certain embodiments, the invention provides cell lines produced from the cells described herein.

In certain embodiments, the present invention provides a method for producing a cell expressing a protein of interest, wherein the cell has at least one desired property that remains consistent over time, the method comprising the steps of:

a) providing a plurality of cells expressing mRNA encoding a protein of interest;

b) individually dispersing cells into a single culture vessel, thereby providing a plurality of isolated cell cultures;

c) culturing the cells under a desired set of culture conditions using an automated cell culture method characterized in that the conditions are each substantially equivalent for an isolated cell culture, the number of cells per isolated cell culture is normalized during this culture, and wherein the isolated cultures are passaged according to the same schedule;

d) Determining at least one desired characteristic of the protein of interest of the isolated cell culture at least 2 times; and

e) identifying an isolated cell culture having the desired characteristics in both assays. In particular embodiments, the cells produced by the methods described herein are differentiated cells. In particular embodiments, the cells produced by the methods described herein are dedifferentiated cells. In particular embodiments, the dedifferentiated cells are stem cells selected from the group consisting of: pluripotent stem cells, pluripotent stem cells (pluripotent stem cells), totipotent stem cells, induced pluripotent stem cells, embryonic stem cells, cancer stem cells, organ-specific stem cells, and tissue-specific stem cells.

In certain embodiments, the present invention provides a method for producing cells expressing a protein of interest, wherein at least one of the cells has a desired property that remains consistent over time, the method comprising the steps of:

a) providing at least two cells expressing RNA encoding a protein of interest;

e) identifying an isolated cell culture having the desired characteristics in both assays. In particular embodiments, the cells produced by the methods described herein are differentiated cells. In particular embodiments, the cells produced by the methods described herein are dedifferentiated cells. In particular embodiments, the dedifferentiated cells are stem cells selected from the group consisting of pluripotent stem cells, multipotent stem cells, totipotent stem cells, induced pluripotent stem cells, embryonic stem cells, cancer stem cells, organ-specific stem cells and tissue-specific stem cells.

In certain embodiments, the plurality of cells in step a) of the methods described herein are cultured for a period of time prior to dispersion in step b).

In certain embodiments, the single culture vessel used in the methods of the invention is selected from the group consisting of: single well of multi-well plate and vial.

In certain embodiments, the method further comprises the step of determining the growth rate of the plurality of isolated cell cultures, and grouping the isolated cell cultures into groups by their growth rate, such that the difference between the fastest and slowest growth rates in any group does not exceed 1, 2, 3, 4, or 5 hours between steps b) and c).

In certain embodiments, the method further comprises the step of preparing a storage stock of the one or more isolated cultures. In certain embodiments, the method further comprises the steps of preparing one or more replicate groups of the isolated cell culture and culturing the one or more replicate groups separately from the cell culture isolated from the source.

In certain embodiments, the assay in step d) of the method of the invention is a functional assay for a protein.

In certain embodiments, the at least one characteristic that remains consistent in step e) is protein function.

In certain embodiments, the culturing in step c) of the method of the invention is a robotic cell incubator. In certain embodiments, the robotic cell incubator comprises a multichannel robotic pipettor. In certain embodiments, the multichannel robotic pipettor comprises at least 96 channels. In certain embodiments, the robotic cell culture apparatus further comprises a preferred-picking arm.

In certain embodiments, the automated process comprises one or more of the following: medium removal, medium replacement, cell washing, reagent addition, cell removal, cell dispersion, and cell passaging.

In certain embodiments, the plurality of isolated cell cultures for use in the present invention is at least 50 cultures. In other embodiments, the plurality of isolated cell cultures is at least 100 cultures. In other embodiments, the plurality of isolated cell cultures is at least 500 cultures. In other embodiments, the plurality of isolated cell cultures is at least 1000 cultures.

In certain embodiments, the growth rate is determined by a method selected from the group consisting of: measurement of ATP, measurement of cell confluence, light scattering, optical density measurement. In certain embodiments, the difference between the fastest and slowest growth rates in the group does not exceed 1, 2, 3, 4, or 5 hours.

In certain embodiments, the culturing in step c) of the methods of the invention is for at least 2 days.

In certain embodiments, the growth rate of a plurality of isolated cell cultures is determined by dispersing cells and measuring cell confluence. In certain embodiments, the cells in each isolated cell culture of the methods of the invention are dispersed prior to measuring cell confluence. In certain embodiments, the dispersing step comprises adding trypsin into the pores and eliminating clots. In certain embodiments, cell confluence is measured for a plurality of separated cell cultures using an automated microplate reader.

In certain embodiments, at least 2 confluency measurements are taken prior to calculating the growth rate. In certain embodiments, cell confluence is measured by an automated plate reader, and the confluence value is used with a software program that calculates the growth rate.

In certain embodiments, the isolated cell culture in step d) is characterized for a desired property selected from the group consisting of: fragility (fragility), morphology, attachment to solid surfaces; lack of attachment to solid surfaces and protein function. In other embodiments, the desired property is UPR, cell viability, ability to increase protein production, yield, folding (folding), assembly, secretion, integration into the cell membrane, post-translational modification or glycosylation, or any combination thereof.

In certain embodiments, the cells used in the methods of the invention are eukaryotic cells. In certain embodiments, the eukaryotic cell used in the methods of the invention is a mammalian cell. In certain embodiments, the mammalian cell line is selected from the group consisting of: NS0 cells, CHO cells, COS cells, HEK-293 cells, HUVEC, 3T3 cells, and HeLa cells. In another embodiment, the mammalian cell line is Perc 6.

In certain embodiments, the protein of interest expressed in the methods of the invention is a human protein. In certain embodiments, the protein of interest is a heteromultimer. In certain embodiments, the protein of interest is a G protein-coupled receptor. In other embodiments, the protein does not have a known ligand. In other embodiments, there are no known assays to detect functional expression of the protein.

In certain embodiments, the methods of the invention further comprise the following steps after the identifying step:

a) expanding a storage aliquot of the cell culture identified in step e) under desired culture conditions; and

b) determining whether the expanded cell culture of a) has the desired characteristic.

In certain embodiments, the invention provides a matched panel of clonal cell lines, wherein the clonal cell lines are of the same cell type, and wherein each cell line in the panel expresses a protein of interest, and wherein the clonal cell lines in the panel are matched to share the same physiological properties, thereby allowing parallel processing.

In certain embodiments, the invention provides a matched panel of clonal cell lines, wherein the clonal cell lines are of the same cell type, and wherein at least two cell lines in the panel express a protein of interest, and wherein the clonal cell lines in the panel are matched to share the same physiological properties, thereby allowing parallel processing.

In certain embodiments, the present invention provides that the combination of clonal cell lines matches a panel of subjects, wherein the clonal cell lines are of the same cell type, and wherein at least two cell lines in the panel express a multi-subunit protein of interest, and wherein the clonal cell lines each comprise a different combination of subunits of the multi-subunit protein of interest; and wherein the clonal cell lines in the subject group are matched such that they are cultured in parallel under the same cell culture conditions.

In certain embodiments, the physiological property is growth rate. In other embodiments, the physiological property is adhesion to the surface of the tissue culture. In other embodiments, the physiological property is a Z' factor. In other embodiments, the physiological property is the expression level of an RNA encoding a protein of interest. In other embodiments, the physiological property is the expression level of the protein of interest. In other embodiments, the physiological property is the level of activity of an RNA encoding a protein of interest. In certain embodiments, the growth rates of the clonal cell lines in a subject group are within 1, 2, 3, 4, or 5 hours of each other. In other embodiments, the culture conditions used to match the subject group are the same for all clonal cell lines within the subject group.

In certain embodiments, the clonal cell line used in the matched panel of subjects is a eukaryotic cell line. In certain embodiments, the eukaryotic cell line is a mammalian cell line. In certain embodiments, the clonal cell line cells used in the matched subject group are selected from the group consisting of: primary cells and immortalized cells.

In certain embodiments, the cell line cells used in the matched subject group are prokaryotic or eukaryotic cells. In certain embodiments, the cell line cells used in the matched subject group are eukaryotic cells and are selected from the group consisting of: fungal cells, insect cells, mammalian cells, yeast cells, algae, crustacean cells, arthropod cells, avian cells, reptilian cells, amphibian cells, and plant cells. In certain embodiments, the cell line cells used in the matched subject group are mammalian and are selected from the group consisting of: humans, non-human primates, bovines, porcines, felines, rats, marsupials, murines, canines, ovines, caprines (caprines), rabbits, guinea pigs, hamsters.

In certain embodiments, cells in cell lines matching the subject panel are engineered to express a protein of interest. In certain embodiments, cells in the cell lines that match the subject panel express a protein of interest from an introduced nucleic acid that encodes the protein, and in the case of multimeric proteins, subunits of the protein. In certain embodiments, the cell expresses a protein of interest from an endogenous nucleic acid, and wherein the cell is engineered to activate transcription of the endogenous protein, or in the case of multimeric proteins, of subunits of the protein.

In certain embodiments, the subject group comprises at least 4 clonal cell lines. In other embodiments, the panel of subjects comprises at least 6 clonal cell lines. In other embodiments, the subject group comprises at least 25 clonal cell lines.

In certain embodiments, 2 or more clonal cell lines in a subject group express the same protein of interest. In other embodiments, 2 or more clonal cell lines in a subject group express different proteins of interest.

In certain embodiments, the cell lines in the subject panel express different forms of the protein of interest, wherein the forms are selected from the group consisting of: isoforms, amino acid sequence variants, splice variants, truncated forms, fusion proteins, chimeras, or combinations thereof. In other embodiments, the form is an active form, a modified form, a glycosylated form, a proteolytic form, or a functional form, or a combination thereof. In other embodiments, the form is selected from the group consisting of: isoforms, amino acid sequence variants, splice variants, truncated forms, fusion proteins, chimeras, active forms, modified forms, glycosylated forms, proteolytic forms, functional forms, or combinations thereof.

In certain embodiments, the cell lines in the subject panel express different proteins in a panel of proteins of interest, wherein the panel of proteins of interest is selected from the group consisting of: proteins in the same signaling pathway, expression libraries of similar proteins, monoclonal antibody heavy chain libraries, monoclonal antibody light chain libraries, and SNPs.

In certain embodiments, the protein of interest expressed in the subject panel is a single chain protein. In certain embodiments, the single-chain protein is a G protein-coupled receptor. In certain embodiments, the G protein-coupled receptor is a taste receptor. In certain embodiments, the taste receptor is selected from the group consisting of: bitter taste receptors, sweet taste receptors, salty taste receptors and umami taste receptors.

In other embodiments, the protein of interest expressed in the subject panel is a multimeric protein. In certain embodiments, the protein is a heterodimer or heteromultimer.

In certain embodiments, the protein of interest expressed in the subject group is selected from the group consisting of: ion channels, G protein-coupled receptors (GPCRs), tyrosine receptor kinases, cytokine receptors, nuclear steroid hormone receptors, and immunoreceptors. In other embodiments, the protein of interest expressed in the subject panel is an antibody or a biologic. In certain embodiments, the protein expressed in the matched panel is an epithelial sodium channel (ENaC). In certain embodiments, the ENaC comprises an alpha subunit, a beta subunit, and a gamma subunit. In other embodiments, the cell lines in the subject group express different ENaC isoforms. In other embodiments, the cell lines in the subject panel comprise different proteolytic isoforms of ENaC. In certain embodiments, the ENaC is human ENaC. In certain embodiments, the protein expressed in the matched panel is a voltage-gated sodium channel (NaV). In certain embodiments, the NaV comprises an alpha subunit and 2 beta subunits. In certain embodiments, the NaV is human NaV.

In certain embodiments, the proteins expressed in the matched panel of subjects are selected from the group consisting of: gamma-aminobutyric acid A receptor (GABA)_AReceptor), gamma-aminobutyric acid B receptor (GABA)_BReceptor) and gamma-aminobutyric acid C receptor (GABA)_cReceptor). In certain embodiments, the proteinThe proton is GABA_AA receptor. In certain embodiments, GABA_AThe receptor comprises 2 alpha subunits, 2 beta subunits, and gamma or delta subunits.

In certain embodiments, clonal cell lines in a subject group are generated simultaneously or within no more than 4 weeks of each other. In other embodiments, the clonal cell lines in a subject group are generated using substantially the same methodology (for isolation, maintenance, or testing of clonal cell lines of a subject group).

In certain embodiments, the invention provides a cell expressing a monomeric protein of interest from an introduced nucleic acid encoding the monomeric protein of interest, characterized in that it produces the protein of interest in a form that is or is capable of becoming biologically active, wherein the cell is cultured in the absence of selective pressure and wherein the expression of the protein does not change by more than 5% within 3 months. In certain embodiments, the expression of the protein does not change by more than 5% within 6 months. In certain embodiments, the monomeric protein of interest does not have a known ligand.

In certain embodiments, the invention provides a cell expressing a monomeric protein of interest from an introduced nucleic acid encoding the monomeric protein of interest, characterized in that it produces the protein of interest in a form that is or is capable of becoming biologically active, wherein the cell is cultured in the absence of selective pressure and wherein the expression of the protein does not change by more than 30% within 3 months. In certain embodiments, the expression of the protein does not change by more than 30% within 6 months.

In certain embodiments, the invention provides a cell that expresses at least one RNA of interest, wherein the RNA of interest is encoded by an introduced nucleic acid, said cell being characterized in that it produces the RNA of interest in a form that is or is capable of becoming biologically active, wherein said cell is cultured in the absence of selective pressure and wherein the expression of the RNA does not change by more than 30% within 3 months. In certain embodiments, the expression of RNA does not change by more than 30% within 6 months.

In certain embodiments, the invention provides a cell expressing a protein of interest, wherein the protein of interest is encoded by an introduced nucleic acid, said cell being characterized in that it produces the protein of interest in a form that is or is capable of becoming biologically active, wherein said cell is cultured in the absence of selective pressure and wherein the expression of the protein does not change by more than 30% within 3 months. In certain embodiments, the expression of the protein does not change by more than 30% within 6 months.

In certain embodiments, the invention provides a cell expressing at least one protein of interest, wherein the protein of interest does not have a known ligand or wherein there is no known assay to detect functional expression of the protein of interest; and wherein the protein of interest does not comprise a protein tag.

In certain embodiments, the present invention provides methods for identifying modulators of a protein of interest, comprising the steps of:

a) contacting a cell according to any of the above cell embodiments with a test compound; and

b) detecting a change in activity of the protein of interest in cells contacted with the test compound as compared to the activity of the protein in cells not contacted with the test compound;

wherein a compound that produces a difference in activity in the presence as compared to the absence is a modulator of the protein of interest.

In another embodiment, the invention provides a modulator identified by the method of the preceding paragraph.

In certain embodiments, the present invention provides a cell expressing at least one protein of interest from an introduced nucleic acid encoding the at least one protein of interest, wherein the at least one protein of interest alters a physiological property of the cell, and wherein the physiological property of the cell does not change by more than 25% within 3 months under constant cell culture conditions.

In certain embodiments, the invention provides a cell that expresses a protein of interest, wherein the cell is engineered to activate transcription of an endogenous nucleic acid encoding the protein of interest, wherein the protein of interest alters a physiological characteristic of the cell, and wherein the physiological characteristic of the cell does not change by more than 25% within 3 months under constant cell culture conditions.

In certain embodiments, the invention provides a cell that expresses an RNA of interest, wherein the RNA of interest is encoded by the introduced nucleic acid, wherein the at least one RNA of interest alters a physiological property of the cell, and wherein the physiological property of the cell does not change by more than 25% within 3 months under constant cell culture conditions.

A cell that expresses at least one protein of interest from an introduced nucleic acid encoding the at least one protein of interest, said cell being characterized in that it produces the protein of interest in a form that is or is capable of becoming biologically active, and wherein said cell consistently and reproducibly expresses at least 500, 2,500, 5,000, or 100,000 picograms of protein per cell per day.

A cell that expresses a protein of interest, wherein the cell is engineered to activate transcription of an endogenous nucleic acid encoding the protein of interest, said cell being characterized in that it produces the protein of interest in a form that is or is capable of becoming biologically active, and wherein the cell consistently and reproducibly expresses at least 500, 2,500, 5,000, or 100,000 picograms of protein per cell per day.

In certain embodiments, the cells are produced in a time period selected from less than 1 week, less than 2 weeks, less than 3 weeks, less than 4 weeks, less than 1 month, less than 2 months, less than 3 months, less than 4 months, less than 5 months, less than 6 months, less than 7 months, less than 8 months, or less than 9 months.

In certain embodiments, the invention provides a cell expressing at least one protein of interest from an introduced nucleic acid encoding the at least one protein of interest, the cell being characterized in that it produces the protein of interest in a form that is or is capable of becoming biologically active, wherein the cell is produced in a time period selected from less than 7 months, less than 8 months, or less than 9 months, and wherein the cell consistently and reproducibly expresses at least 0.5, 1.0, 5.0, or 10g/L of the protein.

In certain embodiments, the invention provides a cell that expresses a protein of interest, wherein the cell is engineered to activate transcription of an endogenous nucleic acid encoding the protein of interest, the cell being characterized in that it produces the protein of interest in a form that is or is capable of becoming biologically active, wherein the cell is produced in a time period selected from less than 7 months, less than 8 months, or less than 9 months, and the cell consistently and reproducibly expresses at least 0.5, 1.0, 5.0, or 10g/L of the protein.

In certain embodiments, the cells are produced in a time period selected from less than 3 months, less than 4 months, or less than 6 months. In certain embodiments, the protein is a monomeric protein. In other embodiments, the protein is a multimeric protein. In certain embodiments, the protein of interest does not comprise a protein tag or the cells are cultured in the absence of selective pressure, or a combination thereof. In certain embodiments, the multimeric protein of interest comprises at least 2, 3, 4, 5, or at least 6 subunits. In certain embodiments, the multimeric protein of interest is selected from the group consisting of: ion channels, G protein-coupled receptors (GPCRs), tyrosine receptor kinases, cytokine receptors, nuclear steroid hormone receptors, antibodies, biologicals, and immunological receptors. In certain embodiments, the multimeric protein of interest is an ion channel and the cell physiological property is selected from the group consisting of membrane potential, UPR, cell viability, ability to increase protein yield, folding assembly, secretion, integration into the cell membrane, post-translational modification, glycosylation, or any combination thereof.

In another embodiment, the invention provides a cell line produced from a cell described herein.

In certain embodiments, the present invention provides a method of identifying a modulator of a protein of interest, comprising the steps of:

a) contacting a cell described herein (e.g., a cell expressing at least one protein or RNA of interest) with a test compound; and

b) detecting a change in activity of the protein of interest in the cell contacted with the test compound as compared to the activity of the protein in the cell not contacted with the test compound;

In certain embodiments, the invention provides a matched panel of cells or clonal cell lines comprising at least 2 cells described herein (e.g., cells expressing at least one protein or RNA of interest) or 2 clonal cell lines described herein (e.g., cell lines generated from cells described herein), wherein the at least 2 cells or at least 2 clonal cell lines are matched such that they are cultured in parallel under the same cell culture conditions.

In certain embodiments, the matched panel comprises at least 10 cells, 10 clonal cell lines and at least 10 cells or 10 clonal cell lines are matched such that they are cultured in parallel under the same cell culture conditions. In other embodiments, the subject group comprises at least 100 cells or at least 100 clonal cell lines and said at least 100 cells or at least 100 clonal cell lines are cultured under the same cell culture conditions.

In certain embodiments, the present invention provides a matched panel of clonal cell lines, wherein the clonal cell lines are of the same type and comprise a first and a second protein of interest; wherein the first protein of interest is the same in each clonal cell line; wherein the second protein of interest is a component of a functional biological pathway; and wherein:

a) said group of subjects comprises at least 5 cell lines;

b) generating the group of subjects within 6 months;

c) the first and second proteins of interest do not have a protein tag;

d) culturing the clonal cell line in the absence of selective pressure; or

e) any combination of a) -d).

In certain embodiments, the first protein of interest is an antibody and the functional biological pathway is a glycosylation pathway.

In certain embodiments, the present invention provides methods for generating an in vitro correlation of a physiological property in vivo, wherein the method comprises:

a) contacting a compound or compounds having a physiological property with a first cell expressing a first protein of interest;

b) determining the effect of the compound or compounds on the first protein in a functional assay;

c) contacting the compound or compounds with a second cell expressing a second protein of interest;

d) Determining the effect of the compound or compounds on the second protein in a functional assay;

wherein the first and second proteins independently i) do not comprise a protein tag; ii) is consistently and reproducibly produced in a form suitable for a functional assay such that the cells have a Z' factor of at least 0.4 in the functional assay, iii) is expressed in cells cultured in the absence of selective pressure, iv) alters the physiological properties of the cells and wherein the physiological properties of the cells do not change by more than 25% within 3 months under constant cell culture conditions; v) is stably expressed in cells cultured in the absence of selection and wherein the expression of the protein does not change by more than 30% within 3 months, vi) is expressed in cells that also express another protein and are cultured in the absence of selection pressure or vii) any combination thereof; and wherein the profiles obtained in steps a) to d) provide an in vitro correlation of physiological properties in vivo.

In certain embodiments, the first and second proteins of interest are independently selected from monomeric or multimeric proteins. In certain embodiments, the multimeric protein comprises at least 2, 3, 4, 5, or 6 subunits. In certain embodiments, the multimeric protein is a heteromultimeric protein. In certain embodiments, the first and second proteins of interest are independently selected from the group consisting of: ENaC, NaV, GABAA, sweet taste receptor, umami taste receptor, bitter taste receptor, CFTR and GCC.

In certain embodiments, the first and second cells are cells within a subject group of cells that also includes at least one other cell; engineering each cell in a subject population of cells to express a different protein and contacting the cell with a compound or compounds; determining the effect of the compound or compounds on each protein expressed in each cell in the subject set of cells in a functional assay; and the activity profile of the compound or compounds in each cell is used to generate an in vitro correlation of physiological properties.

In certain embodiments, each protein is independently selected from a monomeric protein or a multimeric protein. In certain embodiments, the multimeric protein comprises at least 2, 3, 4, 5, or 6 subunits. In certain embodiments, the multimeric protein is a heteromultimeric protein. In certain embodiments, each protein is independently selected from the group consisting of: ENaC, NaV, GABAA, sweet taste receptor, umami taste receptor, bitter taste receptor, CFTR and GCC.

In certain embodiments, the present invention provides a method for predicting a physiological property of a test compound, wherein the method comprises:

a) Contacting a test compound or a plurality of test compounds with a first cell expressing a first protein of interest as described herein above (e.g., a first protein of interest as described in a method for generating an in vitro correlation of physiological properties in vivo);

b) determining the effect of the test compound or compounds on the first protein in a functional assay;

c) contacting the test compound or compounds with a second cell expressing a second protein of interest described above (e.g., a second protein of interest as described in the methods for generating an in vitro correlation of physiological properties in vivo);

d) determining the effect of the test compound or compounds on the second protein in a functional assay;

e) comparing the activity profile of the test compound obtained in steps a) to d) with the in vitro correlation as generated by the method described hereinbefore (e.g. the method for generating an in vitro correlation of a physiological property in vivo),

wherein the first and second proteins independently i) do not comprise a protein tag, ii) are consistently and reproducibly produced in a form suitable for use in a functional assay such that the cell has a Z' factor of at least 0.4 in the functional assay, iii) are expressed in cells cultured in the absence of selective pressure, iv) alter a physiological characteristic of the cell and wherein the physiological characteristic of the cell does not change by more than 25% within 3 months under constant cell culture conditions; v) is stably expressed in cells cultured in the absence of selective pressure and wherein the expression of the protein does not change by more than 30% within 3 months, vi) is expressed in cells that also express another protein and are cultured in the absence of selective pressure or vii) any combination thereof; and wherein the test compound or compounds are predicted to have an in vitro correlated physiological property if the activity profile of the test compound is at least 90% identical to the in vitro correlated activity profile.

In certain embodiments, the present invention provides methods for confirming a physiological property of a test compound or compounds, wherein the method comprises:

e) comparing the activity profile of the test compound or compounds obtained in steps a) to d) with an in vitro correlation as produced by a method as described hereinbefore (e.g. a method for producing an in vitro correlation of a physiological property in vivo), wherein the first and second proteins independently i) do not comprise a protein tag, ii) are consistently and reproducibly produced in a form suitable for use in a functional assay such that the cells have a Z' factor of at least 0.4 in the functional assay, iii) are expressed in cells cultured in the absence of selective pressure, iv) alter the physiological property of the cells and wherein the physiological property of the cells does not change by more than 25% within 3 months under constant cell culture conditions; v) is stably expressed in cells cultured in the absence of selective pressure and wherein the expression of the protein does not change by more than 30% within 3 months, vi) is expressed in cells that also express another protein and are cultured in the absence of selective pressure or vii) any combination thereof; and wherein the test compound is confirmed to have a physiological property if the activity profile of the test compound or compounds is at least 90% identical to the activity profile of the in vitro correlation.

In certain embodiments, the first and second proteins are independently selected from monomeric or multimeric proteins. In certain embodiments, the multimeric protein comprises at least 2, 3, 4, 5, or at least 6 subunits. In certain embodiments, the multimeric protein is a heteromultimeric protein.

In certain embodiments, the first and second cells are cells in a subject group of cells that also includes at least one other cell; engineering each cell in a subject group of cells to express a different protein and contacting the cell with a test compound or test compounds; determining the effect of the test compound or compounds on each protein of interest expressed in each cell in the subject group of cells in a functional assay; and the activity profile of the test compound or compounds in each cell is used to compare with the profile of in vitro correlations.

In certain embodiments, at least one of the first multimeric protein of interest and the second multimeric protein of interest is a heteromeric protein. In certain embodiments, at least one of the first protein of interest and the second protein of interest is a dimeric protein. In other embodiments, at least one of the first protein of interest and the second protein of interest is a trimeric protein. In certain embodiments, the first protein of interest and the second protein of interest are different forms of a multimeric protein. In certain embodiments, the multimeric protein is a GABAA receptor.

In certain embodiments, at least one of the first or second proteins of interest is part of a functional biological pathway. In certain embodiments, the functional biological pathway is selected from the group consisting of: glycosylation, protein synthesis, UPR, ER, ribosomes, mitochondrial activity, RNA synthesis, post-translational modifications, cell signaling, cell growth, and cell death.

In certain embodiments, the physiological property is a therapeutic effect. In certain embodiments, the physiological property is an adverse effect. In certain embodiments, the effect of a compound or compounds on a physiological property is determined using high throughput screening. In certain embodiments, the comparing step described above is performed in a computer system.

In certain embodiments, the present invention provides a computer-implemented method for determining a physiological property of a test compound or a plurality of test compounds, wherein the method comprises:

(a) receiving a first activity profile of the test compound or compounds, wherein the first activity profile is generated by the method described hereinbefore, and wherein the first activity profile provides an in vitro correlation of physiological properties of the test compound or compounds;

(b) Comparing the first activity profile to a plurality of marker activity profiles stored in a database to determine a measure of similarity between the first activity profile and each of the marker activity profiles in the plurality of marker activity profiles, wherein each of the marker activity profiles provides an in vitro correlation of known physiological properties of a respective known compound or a plurality of known compounds;

(c) determining one or more signature activity profiles that are most similar to the first activity profile based on the similarity measure determined in step (b): and

(d) identifying a known physiological property associated with one or more marker activity profiles determined to be most similar to the first activity profile in step (c) as a physiological property of the test compound or compounds; wherein steps (a), (b), (c) and (d) are performed on a computer using suitable programming.

In certain embodiments, one or more marker activity profiles are most similar to the first activity profile if the similarity measure is above a predetermined threshold.

In certain embodiments, the present invention provides a computer-implemented method for characterizing a test compound or a plurality of test compounds as being associated with a particular physiological property, wherein the method comprises:

(b) clustering a plurality of activity profiles, the plurality of activity profiles comprising the first activity profile and a plurality of signature activity profiles, wherein each of the signature activity profiles provides an in vitro correlation to a known physiological characteristic of a respective known compound or a plurality of known compounds;

(c) identifying one or more signature activity profiles of the plurality of signature activity profiles that cluster with the first activity profile; and

(d) characterizing a test compound or compounds as being associated with the known physiological property of each known compound or compounds corresponding to one or more marker activity profiles identified in step (c) clustered with the first activity profile;

wherein steps (a), (b), (c) and (d) are performed on a computer using suitable programming.

In certain embodiments, the present invention provides a computer-implemented method of classifying a test compound or a plurality of test compounds according to a physiological property using a classifier (classifier), wherein the method comprises:

(a) Training a classifier using a plurality of marker activity profiles stored in a database to classify a test compound or a plurality of test compounds according to a pharmacological property, wherein each of said marker activity profiles provides an in vitro correlation to a known physiological property of a respective known compound or a plurality of known compounds; and

(b) processing the first activity profile generated by the method described hereinabove using the classifier to classify the test compound or compounds by physiological properties;

wherein steps (a) and (b) are performed on a computer using suitable programming.

In certain embodiments, the invention provides a computer-implemented method of classifying a test compound or a plurality of test compounds by physiological property using a classifier, wherein the method comprises:

(a) training a classifier using a plurality of signature activity profiles stored in a database to classify a compound or compounds according to pharmacological properties, wherein each of said signature activity profiles provides an in vitro correlation to a known in vivo pharmacological property of the respective compound; and

(b) processing the first activity profile generated by the method described above using the classifier to classify the test compound or compounds according to a physiological property,

(c) Training a classifier using a plurality of signature activity profiles stored in a database to classify a test compound or a plurality of test compounds according to a physiological property, wherein each of said signature activity profiles provides an in vitro correlation to a known physiological property of a respective known compound or plurality of compounds;

(d) wherein steps (a) and (b) are performed on a computer using suitable programming.

In certain embodiments, the invention provides a method for characterizing a combination of active subunits of a multimeric protein of interest in a cell, wherein the method comprises:

(a) contacting a first cell expressing a first subunit of a multimeric protein of interest with a test compound or test compounds;

(b) contacting a second cell expressing a second subunit of the multimeric protein of interest with a test compound or test compounds;

(c) contacting a third cell expressing the first and second subunits of the multimeric protein of interest with a test compound or test compounds;

(d) determining the effect of the test compound or compounds on the multimeric protein (when it is expressed in the first cell, the second cell, the third cell) in a functional assay;

(e) Inferring whether the first and/or second subunit is part of a biologically active multimeric protein; and

wherein the features obtained in steps a) to d) provide an in vitro correlation of physiological properties in vivo, and wherein the first and second subunits of the multimeric protein independently do not comprise a protein tag, are expressed in cells cultured in the absence of selective pressure, or any combination thereof.

In certain embodiments, the multimeric protein of interest is a heterodimer. In other embodiments, the multimeric protein of interest is a heterotrimer.

(c) contacting a third cell expressing a third subunit of the multimeric protein of interest with the test compound or compounds;

(d) contacting a fourth cell expressing the first, second and third subunits of the multimeric protein of interest with a test compound or test compounds;

(e) Determining the effect of the test compound or compounds on the multimeric protein (when it is expressed in the first cell, the second cell, the third cell and the fourth cell) in a functional assay;

(f) inferring whether the first, second and/or third subunit is part of a biologically active multimeric protein;

wherein the first, second, and third subunits of the multimeric protein independently do not comprise a protein tag, are expressed in a cell cultured in the absence of selective pressure, or any combination thereof.

In certain embodiments, the multimeric protein is a heterotrimer. In certain embodiments, the multimeric protein is a GABAA receptor.

In certain embodiments, the invention provides a subject panel of cells, wherein the subject panel comprises a first cell and a second cell, wherein the first and second cells have been engineered to express the same subunit of a multimeric protein of interest, wherein the physiological properties of the multimeric protein of interest in the first cell are different from the physiological properties of the multimeric protein in the second cell, and wherein the first and second cells are derived from the same host cell line; wherein the subunits of the multimeric protein of interest do not comprise a protein tag, are expressed in a cell cultured in the absence of selective pressure, or any combination thereof.

In certain embodiments, the invention provides a subject panel of clonal cell lines, wherein each cell line has been engineered to express the same subunit of a multimeric protein of interest, and wherein the physiological properties of the multimeric protein in each cell line are different from the physiological properties of the multimeric protein of interest in another cell line of the subject panel, and wherein the cell lines in the subject panel of cell lines are derived from the same host cell line; wherein the subunits of the multimeric protein of interest do not comprise a protein tag, are expressed in cells cultured in the absence of selective pressure, or any combination thereof.

In certain embodiments, the subject group comprises 2 cell lines. In certain embodiments, the subject group comprises 5 cell lines. In certain embodiments, the subject group comprises 10 cell lines.

In certain embodiments, the multimeric protein of interest is NaV.

In certain embodiments, the invention provides cells that have been engineered to express all of the constituent proteins of a functional biological pathway.

In certain embodiments, the pathway has at least 5 protein components. In certain embodiments, the cells are cultured in the absence of selective pressure. In certain embodiments, the component proteins of the biological pathway do not comprise a protein tag.

In certain embodiments, the present invention provides a panel of clonal cell lines comprising a plurality of clonal cell lines, wherein each clonal cell line of the plurality of clonal cell lines has been engineered to express a different odorant receptor; wherein the odorant receptor does not comprise a protein tag, or the odorant receptor is consistently and reproducibly produced in a form suitable for use in a functional assay such that the cells have a Z' factor of at least 0.4 in the functional assay, or a clonal cell line is cultured in the absence of selective pressure, or any combination thereof.

In certain embodiments, the plurality of clonal cell lines comprises at least 10 cell lines. In certain embodiments, the different odorant receptors are human odorant receptors or insect odorant receptors.

In certain embodiments, the different human odorant receptors are selected from the group consisting of: OR10A, OR10C, OR10D, OR10G, OR10H, OR10J, OR10K, OR10Q, OR10R, OR10S, OR10T, OR10V, OR10Z, OR11A, OR11G, OR11H7, OR11L, OR12D, OR13A, OR13C, OR13F, OR13G, OR13H, OR13J, OR 1H, OR2J, OR1F, OR2J, OR 1H, OR2D, OR 1H, OR2J, OR 1H, OR2H, OR 1H, OR7, OR11H, OR11L, OR2H, OR 1H, OR14, OR2H, OR 1H, OR2H, OR14, OR 1H, OR2H, OR 1H, OR14, OR2H, OR1L, OR2H, OR 1H, OR2H, OR14, OR 1H, OR14, OR2H, OR 1H, OR2H, OR14, OR 1H, OR1L, OR2H, OR14, OR 1H, OR14, OR2H, OR14, OR1L, OR2H, OR1, OR2J, OR2K, OR2L, OR2V, OR2W, OR2Y, OR2Z, OR3A, OR4B, OR4C, OR4D, OR4E, OR4F, OR4K, OR2L, OR2T, OR2V, OR2W, OR2Y, OR2Z, OR3A, OR4C, OR4D, OR4F, OR4K 52, OR4D, OR4F, OR4D, OR4F, OR4D, OR, OR5AK, OR5AN, OR5AP, OR5AR, OR5AS, OR5AU, OR5B, OR5C, OR5D, OR5F, OR5G, OR5H, OR5I, OR5K, OR5L, OR5M, OR5P, OR5T, OR5V, OR6A, OR6B, OR6C, OR6F, OR6J, OR6K, OR6M, OR6N, OR6P, OR6B, OR6C, OR6F, OR6J, OR6K, OR6M, OR6N, OR6P, OR 7B, OR 7K, OR7C, OR7G 8D, OR 7K, OR7G 8D, OR 7H, OR8H, OR 7H, OR5H, OR6V, OR 6G 7G 8G 7G 8G, OR7G 8G, OR7G 8G, OR7G 8G.

In certain embodiments, the different insect scent receptor is a mosquito scent receptor selected from the group consisting of: IOR100, IOR101, IOR102, IOR103, IOR104, IOR105, IOR106, IOR107, IOR108, IOR109, IOR110, IOR111, IOR112, IOR113, IOR114, IOR115, IOR116, IOR117, IOR118, IOR119, IOR120, IOR121, IOR122, IOR123, IOR124, IOR125, IOR126, IOR127, IOR120, IOR121, IOR122, IOR123, IOR124, IOR125, IOR126, IOR127, IOR, 7119, IOR, 7180, IOR 7180, ORL7119, ORL 7019, ORL 7180, ORL 7019, ORL707, ORL 7019, ORL 7180, ORL 7019, ORL 7180, ORL 7019, ORL707, ORL 7019, ORL 7180, ORL 7019, ORL707, ORL 7019, ORL 7180, ORL 7019, ORL707, ORL 7019, ORL707, ORL 7019, ORL707, ORL 7019, ORL707, ORL 7019, TPR2308, TPR2309, TPR2310, TPR2312, TPR2314, TPR2315, TPR2316, TPR2317, TPR2318, TPR2319, TPR2320, TPR2321, TPR698, TPR699, TPR700, TPR701, TPR702, TPR703, TPR704, TPR705, TPR706, TPR707, TPR708, TPR709, TPR710, TPR711, TPR712, TPR713, TPR714, TPR715, TPR716, TPR771, TPR718, TPR719, TPR720, TPR721, TPR722, TPR723, TPR724, TPR725, TPR726, TPR727, TPR729, TPR717, TPR731, TPR732, TPR734, TPR762, TPR769, TPR 780, TPR739, TP.

In certain embodiments, the present invention provides a method for generating an odor activity profile of a test compound or composition, wherein the method comprises:

i. contacting a subject set described herein (e.g., a subject set comprising a clonal cell line of a plurality of clonal cell lines, wherein each clonal cell line of the plurality of clonal cell lines has been engineered to express a different odorant receptor) with a test compound or composition; and

ii measuring the effect of the test compound or composition on the activity of at least 2 different odorant receptors in the group of subjects in a functional test, wherein the activity measured in step (ii) provides an odorant activity profile of the test compound or composition.

In certain embodiments, the present invention provides a method of identifying a second test compound that mimics the odor of a first test compound or composition, wherein the method comprises:

i. contacting a subject set described herein (e.g., a subject set comprising a clonal cell line of a plurality of clonal cell lines, wherein each clonal cell line of the plurality of clonal cell lines has been engineered to express a different odorant receptor) with a second test compound;

ii testing the effect of the second test compound on the activity of at least 2 odorant receptors in the group of subjects in a functional assay;

(iii) comparing the odor activity profile of the second test compound obtained in step (ii) with the odor activity profile of the first test compound or composition; wherein the second test compound mimics the odor of the first test compound or composition if the odor activity spectrum of the second test compound is similar to the odor activity spectrum of the first test compound or composition.

In certain embodiments, the present invention provides a method of identifying a second test compound that alters the odor activity profile of a first test compound or composition, wherein the method comprises:

i. generating an odor activity profile of a second test compound in the presence of a first test compound or composition according to methods described herein (e.g., methods for generating an odor activity profile of a test compound or composition);

ii comparing the odor activity profile obtained in step (i) with the odor activity profile of the first test compound or composition in the absence of the second test compound; wherein the second test compound alters the odor activity spectrum of the first test compound or composition if the odor activity spectrum of the first test compound or composition alone is different from the odor activity spectrum of the second test compound in the presence of the first test compound or composition.

In certain embodiments, the invention provides a computer-implemented method for identifying an odor associated with a test compound, wherein the method comprises:

(a) receiving a first odor activity profile of a test compound, wherein the first odor activity profile is produced by a method described herein (e.g., a method for producing an odor activity profile of a test compound or composition);

(b) comparing the first odor activity profile to a plurality of hallmark odor activity profiles stored in a database to determine a measure of similarity between the first odor activity profile and each of the hallmark odor activity profiles in the plurality of hallmark odor activity profiles, wherein each of the hallmark odor activity profiles corresponds to a respective known compound having a known odor, and wherein each of the hallmark odor activity profiles is generated by a method described herein (e.g., a method for generating an odor activity profile of a test compound or composition);

(c) determining one or more hallmark odor activity profiles that are most similar to the first odor activity profile based on the similarity measure determined in step (b); and

(d) identifying an odor associated with one or more hallmark odor activity profiles determined to be most similar to the first odor activity profile in step (c) as an odor associated with the known compound;

In certain embodiments, one or more hallmark odor activity profiles are most similar to the first odor activity profile if the similarity measure is above a predetermined threshold.

In certain embodiments, the present invention provides a computer-implemented method for characterizing a compound as being associated with a particular odor, wherein the method comprises:

(a) receiving a first odor activity profile of the compound, wherein the first odor activity profile is produced by a method described herein (e.g., a method for producing an odor activity profile of a test compound or composition);

(b) clustering a plurality of odor activity profiles, the plurality of odor activity profiles comprising the first odor activity profile and a plurality of hallmark odor activity profiles, wherein each of the hallmark odor activity profiles corresponds to a respective known compound having a known odor, and wherein the hallmark odor activity profiles are generated by a method described herein (e.g., a method for generating an odor activity profile of a test compound or composition);

(c) identifying one or more hallmark odor activity profiles of the plurality of hallmark odor activity profiles clustered with a first odor activity profile; and

(d) Characterizing compounds as being associated with the known odors associated with each compound corresponding to one or more hallmark odor activity profiles identified in step (c) as clustered with the first odor activity profile;

In certain embodiments, the present invention provides a computer-implemented method of classifying a test compound as having an odor using a classifier, wherein the method comprises:

(a) training a classifier using a plurality of hallmark odor activity profiles stored in a database to classify a test compound according to odor, wherein each of the hallmark odor activity profiles corresponds to a known compound each having a known odor, and wherein each of the hallmark odor activity profiles is generated by a method described herein (e.g., a method for generating an odor activity profile of a test compound or composition); and

(b) processing a first odor activity profile of a test compound or composition produced by a method described herein (e.g., a method for producing an odor activity profile of the compound) using the classifier to classify the compound according to a known odor;

Performing steps (a) and (b) on a computer using suitable programming.

processing a first odor activity profile of a test compound or composition produced by a method described herein (e.g., a method for producing an odor activity profile of the compound) using the classifier to classify the compound according to a known odor, wherein the classifier is trained according to a method comprising:

training a classifier using a plurality of hallmark odor activity profiles stored in a database to classify a test compound according to odor, wherein each of the hallmark odor activity profiles corresponds to a known compound each having a known odor, and wherein each of the hallmark odor activity profiles is generated by a method described herein (e.g., a method for generating an odor activity profile of a test compound or composition);

where the processing is performed on a computer using suitable programming.

In certain embodiments, the present invention provides a computer-implemented method for correlating one or more test compounds with odor, wherein the method comprises:

(a) Accepting a first odor activity profile of a first test compound, wherein the first odor activity profile is produced by a method described herein (e.g., a method for producing an odor activity profile of a test compound or composition), and wherein the first test compound has a known odor;

(b) comparing the first odor activity profile to a plurality of hallmark odor activity profiles stored in a database to determine a measure of similarity between the first odor activity profile and each of the hallmark odor activity profiles in the plurality of hallmark odor activity profiles, wherein each of the hallmark odor activity profiles corresponds to a respective known compound, and wherein each of the hallmark odor activity profiles is generated by a method described herein (e.g., a method for generating an odor activity profile of a test compound or composition);

(c) determining one or more hallmark odor activity profiles that are most similar to the first odor activity profile based on the similarity measure determined in step (b);

(d) characterizing each test compound corresponding to the one or more hallmark odor activity profiles determined to be most similar to the first odor activity profile in step (c) as being associated with the known odor;

In certain embodiments, the one or more hallmark odor activity profiles are most similar to the first odor activity profile if the similarity measure is above a predetermined threshold.

In certain embodiments, the present invention provides a computer-implemented method for characterizing one or more test compounds as being associated with a particular odor, wherein the method comprises:

(a) accepting a first odor activity profile of a first test compound, wherein the first odor activity profile is produced by a method described herein (e.g., a method for producing an odor activity profile of a test compound or composition), and the first test compound has a known odor;

(b) clustering a plurality of odor activity profiles, the plurality of odor activity profiles comprising the first odor activity profile and a plurality of hallmark odor activity profiles, wherein each of the hallmark odor activity profiles corresponds to a respective known compound, and wherein each of the hallmark odor activity profiles is generated by a method described herein (e.g., a method for generating an odor activity profile of a test compound or composition);

(d) characterizing the compound as being associated with the known odor associated with each compound corresponding to one or more hallmark odor activity profiles identified in step (c) as clustered with the first odor activity profile;

In certain embodiments, the present invention provides a computer-implemented method of classifying one or more test compounds as having an odor using a classifier, wherein the method comprises:

processing a first odor activity profile generated by a method described herein (e.g., a method for generating an odor activity profile of a test compound or composition) using the classifier, wherein the first odor activity profile corresponds to a first test compound having a known odor, to classify one or more hallmark odor activity profiles of a plurality of hallmark odor activity profiles stored in a database as having the known odor, wherein the classifier is trained according to a method comprising:

Training a classifier using the plurality of hallmark odor activity profiles to classify the one or more hallmark odor activity profiles as having an odor, wherein each of the hallmark odor activity profiles corresponds to a respective known compound, and wherein each of the hallmark odor activity profiles is generated by a method described herein (e.g., a method for generating an odor activity profile of a test compound or composition);

where the processing is performed on a computer using suitable programming.

In certain embodiments, the RNA of interest is an siRNA or an antisense RNA. In certain embodiments, the protein of interest comprises at least 2, 3, 4, 5, or 6 subunits.

In certain embodiments, the protein of interest is an orphan receptor identified by a human gene symbol shown in table 8, selected from the group consisting of: BRS3, GPR42P, FPRL2, GPR81, OPN3, GPR52, GPR21, GPR78, GPR26, GPR37, GPR37L1, GPR63, GPR45, GPR83, GRCAe, GPR153, P2RY5, P2 MAS 10, GPR174, GPR142, GPR139, ADMR, CMKOR1, LGR 1, GPR 36173, CCRL 1, MAS 11, MRGPRGPRGPR, MRGPRF, GPRG 31, MRGX 41, MR3672, GPR1, GPR31, TRAR 1, GPR 3636363636363672, GPR 3636363636363636363636363672, GPR1, GPR 36363672, GPR 363672, GPR 36363636363672, GPR 36363636363636363672, GPR1, GPR 36363672, GPR 36363636363636363672, GPR 3636363672, GPR 36363672, GPR1, GPR 3636363636363672, GPR 363636363636363636363672, GPR 363672, GPR 36363672, GPR 363672, GPR 363636363636363636363672, GPR1, GPR 36363636363672, GPR 363672, GPR1, GPR 3636363636363672, GPR1, GPR 36363672, GPR1, GPR 36363636363636363672, GPR 3636363636363636363672, GPR 3636363636363636363636363636.

In certain embodiments, at least one subunit of the protein of interest is expressed by gene activation. In other embodiments, at least one subunit of the protein of interest is expressed from the introduced nucleic acid.

In certain embodiments, the invention provides a method for producing a cell line, wherein the method comprises culturing a plurality of cell lines in a plurality of parallel cultures under the same culture conditions, and identifying a cell line having at least one property that remains consistent over time.

In certain embodiments, the plurality of parallel cultures comprises at least 50 cell cultures. In other embodiments, the plurality of parallel cultures comprises at least 100 cell cultures. In other embodiments, the plurality of parallel cultures comprises at least 200 cell cultures.

In certain embodiments, the invention provides a protein or proteins that are in vitro correlated with a protein or proteins of interest in vivo, wherein the in vitro correlation is predictive of the function or activity of the corresponding protein or proteins of interest expressed in vivo; wherein the in vitro correlation is a biologically active protein or proteins expressed under in vitro non-physiological conditions; wherein the in vitro correlation comprises at least one functional or pharmacological or physiological property corresponding to the protein or proteins of interest in vivo; and wherein at least 10% of the compounds identified in the high throughput screening using the in vitro correlation are capable of having an in vivo therapeutic effect.

In certain embodiments, the in vitro correlation comprises at least 2, 3, 4, 5, or 6 subunits. In certain embodiments, the at least one protein of in vitro relevance comprises at least 2, 3, 4, 5, or 6 subunits. In certain embodiments, the in vitro correlation comprises a heteromultimer. In certain embodiments, the at least one protein of in vitro relevance comprises a heteromultimer. In certain embodiments, the protein or proteins of in vitro relevance do not comprise a protein tag.

In certain embodiments, the in vitro correlation is stably expressed in cells cultured in the absence of selective pressure. In certain embodiments, the in vitro correlation is expressed in a cell line without causing cytotoxicity. In certain embodiments, the in vitro correlation is expressed in a cell that does not endogenously express the protein or proteins.

In certain embodiments, a protein or proteins may be produced by a cell of the invention.

In certain embodiments, the invention provides a cell expressing a protein or proteins as described above.

In certain embodiments, the invention provides cell lines produced from cells expressing a protein or proteins as described herein above.

In certain embodiments, the present invention provides methods for identifying modulators of a protein of interest in vivo, comprising the steps of:

a) contacting a cell expressing a protein or proteins as described herein above with a test compound; and

b) detecting a change in activity of the in vitro relevant protein or proteins in cells contacted with the test compound as compared to the activity of the in vitro relevant protein or proteins in cells not contacted with the test compound;

wherein a compound that produces a difference in activity in the presence as compared to the absence is a modulator of the protein of interest in vivo.

In certain embodiments, the present invention provides modulators identified by the methods described in the preceding paragraphs.

In certain embodiments, the cell described in any of the above paragraphs is a differentiated cell. In certain embodiments, the cell described in any of the above paragraphs is a dedifferentiated cell. In other embodiments, the dedifferentiated cells are stem cells selected from the group consisting of: pluripotent stem cells, multipotent stem cells, totipotent stem cells, induced pluripotent stem cells, embryonic stem cells, cancer stem cells, organ-specific stem cells, and tissue-specific stem cells.

In a specific embodiment, the present invention provides a method for producing stem cells, comprising the steps of: dedifferentiating the differentiated cell into a stem cell, wherein the differentiated cell is a cell described herein or a cell produced by a method described herein. In particular embodiments, the stem cell is selected from the group consisting of a pluripotent stem cell, a multipotent stem cell, a totipotent stem cell, an induced pluripotent stem cell, an embryonic stem cell, a cancer stem cell, an organ-specific stem cell, and a tissue-specific stem cell.

In certain embodiments, the present invention provides methods for producing redifferentiated cells, comprising the steps of:

a) dedifferentiating cells described in any of the above paragraphs or cells produced by the methods described herein to produce stem cells; and

b) redifferentiating the stem cells to produce redifferentiated cells. In specific embodiments, the stem cell is selected from the group consisting of: pluripotent stem cells, multipotent stem cells, totipotent stem cells, induced pluripotent stem cells, embryonic stem cells, cancer stem cells, organ-specific stem cells, and tissue-specific stem cells. In certain embodiments, the redifferentiated cells are of a different type than differentiated cells that have not undergone dedifferentiation.

In certain embodiments, the present invention provides methods for producing a non-human organism, comprising the steps of:

(a) dedifferentiating a differentiated cell described herein or a differentiated cell produced by a method described herein to produce a stem cell, wherein the stem cell is an embryonic stem cell or an induced pluripotent stem cell; and

(b) redifferentiating the stem cells to produce the non-human organism. In particular embodiments of such methods, the organism is a mammal. In other particular embodiments of such methods, the mammal is a mouse.

In other aspects, the invention provides redifferentiated cells produced by the methods described herein.

In certain aspects, the invention provides non-human organisms produced by the methods described herein. In certain embodiments of such methods, the organism is a mammal. In particular embodiments of such methods, the mammal is a mouse.

In certain embodiments, cells endogenously expressing a protein of interest can be isolated from a population of cells as described herein. Such isolated cells can be used in the methods and compositions disclosed herein, such as screening methods and groups of subjects.

In certain aspects, provided herein are cells or cell lines stably expressing a sweet taste receptor comprising a sweet taste receptor T1R2 subunit and a sweet taste receptor T1R3 subunit, the expression of at least one of the subunits resulting from the introduction of a nucleic acid encoding the subunit into a host cell, or the activation of a gene encoding the subunit already present in the host cell, the cells or cell lines being derived from the host cell. Optionally, the cell or cell line may also be engineered to produce a G protein.

In particular embodiments, at least one sweet taste receptor subunit is expressed from a nucleic acid encoding the subunit that is introduced into the host cell. In other specific embodiments, at least one sweet taste receptor subunit is expressed from a nucleic acid present in the host cell by gene activation. In other specific embodiments, the host cell: a) is a eukaryotic cell; b) is a mammalian cell; c) at least one subunit or G protein that does not endogenously express a sweet taste receptor; or d) any combination of (a), (b), and (c). In other specific embodiments, the host cell is a HEK-293 cell. In other specific embodiments, the sweet taste receptor a) is a mammal; b) is a human; c) including subunits from different species; d) including as one or more chimeric subunits; or e) any combination of (a) - (d). In other specific embodiments, the sweet taste receptor is functional. In other specific embodiments, the cells or cell lines described herein have a Z' value of at least 0.3 in the assay. In other specific embodiments, the cells or cell lines described herein have a Z' value of at least 0.7 in the assay. In other specific embodiments, the cells or cell lines described herein stably express a sweet taste receptor in culture in the absence of selective pressure. In other specific embodiments, the sweet T1R2 receptor subunit is selected from the group consisting of:

a) Comprises the amino acid sequence of SEQ ID NO: 34 or a corresponding amino acid sequence of another species;

b) comprises a nucleotide sequence substantially identical to SEQ ID NO: 34 or a corresponding amino acid sequence of another species having at least 85% identity;

c) comprising a polypeptide consisting of a sequence that hybridizes under stringent conditions to SEQ ID NO: 31 or encoding SEQ id no: 34 or a nucleic acid that hybridizes to a nucleic acid of a corresponding amino acid sequence of another species; and

d) comprises a nucleotide sequence consisting of a nucleotide sequence identical to SEQ ID NO: 31 or encoding SEQ ID NO: 34 or a nucleic acid of a corresponding amino acid sequence of another species having at least 85% identity to a nucleic acid encoding the amino acid sequence.

In particular embodiments, the cell or sweet taste receptor subunit of a cell T1R2 described herein is encoded by a nucleic acid selected from the group consisting of:

a) comprises the amino acid sequence of SEQ ID NO: 31 of the nucleic acid sequence

b) Comprising encoding a polypeptide comprising SEQ ID NO: 34 or a nucleotide sequence of a polypeptide of the corresponding amino acid sequence of another species;

c) a nucleic acid comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid of a) or b); and

d) Comprises a nucleotide sequence substantially identical to SEQ ID NO: 31 or encoding SEQ ID NO: 34 or a nucleic acid of a nucleotide sequence having at least 95% identity to a nucleic acid of the corresponding amino acid sequence of another species. In other specific embodiments, the sweet taste receptor subunit T1R3 is selected from the group consisting of:

e) comprises the amino acid sequence of SEQ ID NO: 35 or a corresponding amino acid sequence of another species;

f) comprises a nucleotide sequence substantially identical to SEQ ID NO: 35 or a corresponding amino acid sequence of another species has at least 85% identity to the corresponding amino acid sequence of the other species;

g) comprising a polypeptide consisting of a sequence that hybridizes under stringent conditions to SEQ ID NO: 32 or encoding SEQ id no: 35 or a nucleic acid that hybridizes to a nucleic acid of a corresponding amino acid sequence of another species; and

h) h) comprises a sequence consisting of SEQ ID NO: 32 or encoding SEQ ID NO: 35 or a nucleic acid of a corresponding amino acid sequence of another species has at least 85% identity to a nucleic acid encoding the amino acid sequence.

In other specific embodiments, the sweet taste receptor T1R3 subunit is encoded by a nucleic acid selected from the group consisting of:

a) comprises the amino acid sequence of SEQ ID NO: 32;

b) Comprising encoding a polypeptide comprising SEQ ID NO: 35 or a nucleotide sequence of a polypeptide of the corresponding amino acid sequence of another species;

d) comprises a nucleotide sequence substantially identical to SEQ ID NO: 32 or encoding SEQ ID NO: 35 or a nucleic acid of a nucleotide sequence having at least 85% identity to a nucleic acid of a corresponding amino acid sequence of another species.

In other specific embodiments, the G protein is selected from the group consisting of:

a) comprises the amino acid sequence of SEQ ID NO: 36 or 37 or the corresponding amino acid sequence of another species;

b) comprises a nucleotide sequence substantially identical to SEQ ID NO: 36 or 37 or another species or an amino acid sequence having at least 85% identity to the corresponding amino acid sequence;

c) comprising a polypeptide consisting of a sequence that hybridizes under stringent conditions to SEQ ID NO: 33 or encoding SEQ id no: 36 or 37 or a nucleic acid that hybridizes to a nucleic acid of the corresponding amino acid sequence of another species; and

d) comprises a nucleotide sequence consisting of a nucleotide sequence identical to SEQ ID NO: 33 or encoding SEQ ID NO: 36 or 37 or a nucleic acid sequence encoding an amino acid sequence having at least 85% identity to a nucleic acid of the corresponding amino acid sequence of another species.

In other specific embodiments, the G protein is encoded by a nucleic acid selected from the group consisting of:

a) comprises the amino acid sequence of SEQ ID NO: 33;

b) comprises a nucleotide sequence encoding SEQ ID NO: 36 or 37 or a corresponding amino acid sequence of another species;

c) nucleic acids comprising a nucleotide sequence which hybridizes under stringent conditions to the nucleic acid sequence of a) or b);

d) comprises a nucleotide sequence substantially identical to SEQ ID NO: 33 or encoding SEQ ID NO: 36 or 37 or a nucleotide sequence having at least 85% sequence identity to a nucleic acid sequence of the corresponding amino acid sequence of another species.

In certain aspects, provided herein are cells or cell lines stably expressing an umami receptor comprising an umami receptor T1R1 subunit and an umami receptor T1R3 subunit, expression of at least one of the subunits resulting from introduction of a nucleic acid encoding the subunit into a host cell or activation of a gene encoding a nucleic acid encoding the subunit already present in a host cell, the cells or cell lines being derived from a host cell. Optionally, the cell or cell line may also be engineered to produce a G protein.

In a specific embodiment, at least one umami receptor subunit is expressed from a nucleic acid encoding the subunit introduced into the host cell. In other specific embodiments, at least one umami receptor subunit is expressed from a nucleic acid present in the host cell by gene activation. In other specific embodiments, the host cell: a) is a eukaryotic cell; b) is a mammalian cell; c) at least one subunit or G protein that does not endogenously express the umami receptor; or d) any combination of (a), (b), and (c). In other specific embodiments, the host cell is a human HEK-293 cell. In other specific embodiments, the umami receptor a) is a mammalian umami receptor; b) is a human umami receptor; c) comprise subunits from different species; d) comprises one or more subunits that are chimeras; or e) any combination of (a) - (d). In other specific embodiments, the umami receptor is functional. In other specific embodiments, the cells or cell lines described herein have a Z' value of at least 0.3 in the assay. In other specific embodiments, the cells or cell lines described herein have a Z' value of at least 0.7 in the assay. In other embodiments, the cells or cell lines described herein stably express the umami receptor in culture in the absence of selective pressure. In other specific embodiments, the T1R1 receptor subunit of the umami receptor is selected from the group consisting of:

a) Comprises the amino acid sequence of SEQ ID NO: 42-45 or a corresponding amino acid sequence of another species;

b) comprises a nucleotide sequence substantially identical to SEQ ID NO: 42-45 or a corresponding amino acid sequence of another species having at least 85% identity;

c) comprising a polypeptide consisting of a sequence that hybridizes under stringent conditions to SEQ ID NO: 41 or encoding SEQ ID NO: 42-45 or a corresponding amino acid sequence of another species, or a nucleic acid hybridizing to a nucleic acid of the corresponding amino acid sequence of another species; and

d) comprises a nucleotide sequence consisting of a nucleotide sequence identical to SEQ ID NO: 31 or encoding SEQ ID NO: 42-45 or a nucleic acid of a corresponding amino acid sequence of another species, has at least 85% identity to a nucleic acid of the other species.

In particular embodiments, the cells or the umami receptor subunit of cells T1R1 described herein are encoded by a nucleic acid selected from the group consisting of:

a) comprises the amino acid sequence of SEQ ID NO: 41: the nucleic acid of (1);

b) comprising encoding a polypeptide comprising SEQ ID NO: 42-45 or a nucleotide sequence of a polypeptide of the corresponding amino acid sequence of another species;

d) comprises a nucleotide sequence substantially identical to SEQ ID NO: 31 or encoding SEQ ID NO: 34 or a nucleic acid of a nucleotide sequence having at least 95% identity to a nucleic acid of the corresponding amino acid sequence of another species. In other specific embodiments, the umami receptor subunit T1R3 is selected from the group consisting of:

f) comprises a nucleotide sequence substantially identical to SEQ ID NO: 35 or a corresponding amino acid sequence of another species has at least 85% identity to the umami receptor subunit;

h) comprises a nucleotide sequence consisting of a nucleotide sequence identical to SEQ ID NO: 32 or encoding SEQ ID NO: 35 or a nucleic acid of a corresponding amino acid sequence of another species has at least 85% identity to a nucleic acid encoding the amino acid sequence.

In other specific embodiments, the umami receptor T1R3 subunit is encoded by a nucleic acid selected from the group consisting of:

a) Comprises the amino acid sequence of SEQ ID NO: 32;

a) comprises the amino acid sequence of SEQ ID NO: 33;

c) nucleic acid comprising a nucleotide sequence which hybridizes under stringent conditions to the nucleic acid sequence of a) or b);

d) comprises a nucleotide sequence substantially identical to SEQ ID NO: 33 or encoding SEQ ID NO: 36 or 37 or a nucleotide sequence having at least 95% sequence identity to a nucleic acid sequence of the corresponding amino acid sequence of another species.

In one embodiment, the cells and cell lines of the invention produce functional and physiologically relevant umami or sweet taste receptors. They are therefore useful for identifying and selecting modulators of umami or sweet taste receptors.

In another embodiment, the cells and cell lines of the invention stably express umami or sweet taste receptors for 1 to 4 weeks, 1 to 9 months, or any time in between.

In another embodiment, the cells and cell lines of the invention express the umami receptor or the sweet taste receptor at substantially the same level for 1 to 4 weeks, 1 to 9 months, or any time therebetween.

In a specific embodiment, the expression level is measured using a functional assay.

In other embodiments, the invention relates to modulators of umami or sweet taste receptors identified using the cells and cell lines of the invention, and the use of such modulators in modifying the taste of products, including foods and pharmaceuticals, or in the treatment of diseases in which umami or sweet taste receptors are involved, including diabetes and obesity.

In certain embodiments, provided herein are methods for producing a cell or cell line described herein (e.g., a cell or cell line stably expressing a sweet taste receptor), comprising the steps of:

a) introducing into a host cell a first vector comprising a nucleic acid encoding a sweet taste receptor T1R2 subunit, a second vector comprising a nucleic acid encoding a sweet taste receptor T1R3 subunit, and optionally a third vector comprising a nucleic acid encoding a G protein;

b) introducing a first molecular beacon detecting expression of the sweet taste receptor T1R2 subunit, a second molecular beacon detecting expression of the sweet taste receptor T1R3 subunit, and optionally a third molecular beacon detecting expression of a G protein into the host cell produced in step a); and

c) isolating cells expressing the T1R2 subunit, the T1R3 subunit, and optionally the G protein.

In certain embodiments, provided herein are methods for producing a cell or cell line described herein (e.g., a cell or cell line stably expressing an umami receptor), comprising the steps of:

a) introducing into a host cell a first vector comprising a nucleic acid encoding an umami receptor T1R1 subunit, a second vector comprising a nucleic acid encoding an umami receptor T1R3 subunit, and a third vector comprising a nucleic acid encoding a G protein;

b) introducing a first molecular beacon detecting expression of the umami receptor T1R1 subunit, a second molecular beacon detecting expression of the umami receptor T1R3 subunit and optionally a third molecular beacon detecting expression of a G protein into the host cell produced in step a); and

c) isolating cells expressing the T1R1 subunit, the T1R3 subunit, and optionally the G protein.

In particular embodiments, the methods described herein (e.g., methods for producing a cell or cell line stably expressing a sweet or umami receptor) further comprise the step of producing a cell line from the cell isolated in step c). In other specific embodiments, the host cell:

a) is a eukaryotic cell;

b) is a mammalian cell;

c) at least one subunit or G protein that is not endogenously expressed to the sweet taste receptor or the umami taste receptor; or

d) any combination of a), b), and c).

In other embodiments, the methods described herein (e.g., methods for producing a cell or cell line stably expressing a sweet or umami receptor) further comprise the steps of:

a) culturing the cells for a period selected from 1 to 4 weeks, 1 to 9 months, or any time therebetween;

b) periodically measuring the expression of the sweet or umami receptor or subunit thereof during these time periods, measuring the expression at the RNA or protein level; and

c) selecting a cell or cell line characterized by substantially stable expression of a sweet or umami receptor or subunit thereof over a period of time selected from 1 to 4 weeks, 1 to 9 months, or any time therebetween.

In other embodiments, the methods described herein (e.g., methods for producing a cell or cell line stably expressing a sweet taste receptor or an umami taste receptor) further comprise the steps of:

In certain embodiments, measurement of the protein expression level of a sweet taste receptor is performed using a functional assay. In certain embodiments, the separating step utilizes a fluorescence activated cell sorter (Beckman Coulter, Miami, FL).

In particular embodiments, provided herein are methods for identifying modulators of sweet taste receptor function, comprising the steps of contacting at least one cell or cell line stably expressing a sweet taste receptor described herein with at least one test compound and detecting a change in sweet taste receptor function. In particular embodiments, the modulator is selected from the group consisting of: a sweet taste receptor inhibitor, a sweet taste receptor antagonist, a sweet taste receptor blocker, a sweet taste receptor activator, a sweet taste receptor agonist, or a sweet taste receptor enhancer. In other specific embodiments, the sweet taste receptor is a human sweet taste receptor. In other specific embodiments, the test compound is a small molecule, chemical moiety, polypeptide, or antibody. In other specific compounds, the test compound is a library of compounds. In other specific embodiments, the library is a small molecule library, a combinatorial library, a peptide library, or an antibody library. In particular embodiments, the modulators are selective for enzymatically modified forms of sweet taste receptors.

In a specific embodiment, provided herein is a method for identifying a modulator of umami receptor function, comprising the steps of contacting at least one cell or cell line stably expressing an umami receptor described herein with at least one test compound and detecting a change in umami receptor function. In particular embodiments, the modulator is selected from the group consisting of: an umami receptor inhibitor, an umami receptor antagonist, an umami receptor blocker, an umami receptor activator, an umami receptor agonist, or an umami receptor potentiator. In other specific embodiments, the umami receptor is a human umami receptor. In other specific embodiments, the test compound is a small molecule, chemical moiety, polypeptide, or antibody. In other specific compounds, the test compound is a library of compounds. In other specific embodiments, the library is a small molecule library, a combinatorial library, a peptide library, or an antibody library. In particular embodiments, the modulator is selective for enzymatically modified forms of the umami receptor.

In particular embodiments, provided herein are modulators identified by the methods described herein (e.g., methods for identifying modulators of sweet taste receptor function or umami receptor function).

In particular embodiments, provided herein are cells or cell lines described herein (e.g., cells or cell lines stably expressing a sweet taste receptor or an umami taste receptor) produced by the methods described herein for producing such cells or cell lines. In particular embodiments of such methods, the cells of such methods (e.g., cells stably expressing a sweet taste receptor or an umami taste receptor) have at least one desired property that remains consistent over time, and such methods comprise the steps of:

(a) providing a plurality of cells expressing mRNA encoding a taste receptor (e.g., a sweet taste receptor or an umami taste receptor) and optionally a G protein;

(b) individually dispersing cells into a single culture vessel, thereby providing a plurality of isolated cell cultures;

(c) culturing the cells under a desired set of culture conditions using an automated cell culture process characterized by substantially identical conditions for each isolated cell culture, during which the number of cells per well in each cell culture is normalized, and wherein the isolated cultures are passaged according to the same schedule;

(d) Determining at least one desired characteristic of a taste receptor (e.g., a sweet taste receptor or an umami taste receptor) or of a cell producing the receptor of the isolated cell culture at least 2 times; and

(e) identifying an isolated cell culture having the desired characteristics in both assays. In certain aspects, provided herein are cells or cell lines that produce taste receptors (e.g., sweet or umami receptors) and have at least one desired property that remains consistent over time, the cells or cell lines produced by such methods.

In certain embodiments, cells endogenously expressing a sweet taste receptor, an umami taste receptor, and/or a G protein can be isolated from a population of cells as described herein. Such isolated cells can be used with the methods and compositions described herein (e.g., screening groups of subjects for methods and compositions).

According to one aspect of the invention, the cell or cell line is engineered to stably express a bitter taste receptor. In certain embodiments, the bitter taste receptor is expressed from a nucleic acid introduced into the cell or cell line. In some other embodiments, expression of the bitter taste receptor from an endogenous nucleic acid is activated by engineering the gene. In certain embodiments, the cell or cell line stably expresses at least one other bitter taste receptor. In certain embodiments, at least one additional bitter taste receptor is endogenously expressed. In some other embodiments, at least one additional bitter taste receptor is expressed from a nucleic acid introduced into the cell or cell line. In some other embodiments, the bitter taste receptor and at least one other bitter taste receptor are expressed from an isolated nucleic acid introduced into the cell or cell line. In some other embodiments, the bitter taste receptor and the at least one other bitter taste receptor are both expressed from a single nucleic acid introduced into the cell or cell line.

In certain embodiments, the cell or cell line stably expresses the endogenous G protein. In some other embodiments, the cell or cell line stably expresses the heterologous G protein. In some other embodiments, the cell or cell line stably expresses both the endogenous G protein and the heterologous G protein. In certain embodiments, the G protein is a heteromultimeric G protein comprising 3 different subunits. In certain embodiments, at least one subunit of a heteromultimeric G protein is expressed from a nucleic acid introduced into a cell or cell line. In some other embodiments, at least 2 different subunits are derived from different nucleic acids introduced into the cell or cell line. In some other embodiments, at least 2 different subunits are expressed from the same nucleic acid introduced into the cell or cell line. In some other embodiments, each of the 3 different subunits are expressed from the same nucleic acid introduced into the cell or cell line.

In certain embodiments, the cells in the cell line are eukaryotic cells. In some other embodiments, the cell in the cell line is a mammalian cell. In some other embodiments, the cells in the cell line do not express endogenous bitter taste receptors. In some other embodiments, the cell in the cell line is a eukaryotic cell of a cell type that does not express an endogenous bitter taste receptor. In some other embodiments, the cell in the cell line is a mammalian cell of a cell type that does not express an endogenous bitter taste receptor. In certain embodiments, the cell in the cell line is a human embryonic kidney 293T cell.

In certain embodiments, the bitter taste receptor is a mammal. In some other embodiments, the bitter taste receptor is a human. In some other embodiments, the bitter taste receptor does not have a polypeptide tag at its amino-terminus or carboxy-terminus. In some other embodiments, the bitter taste receptor is a mammalian bitter taste receptor that does not have a polypeptide tag at its amino-terminus or carboxy-terminus. In some other embodiments, the bitter taste receptor is a human bitter taste receptor that does not have a polypeptide tag at its amino-terminus or carboxy-terminus.

In certain embodiments, the cell or cell line produces a Z' value of at least 0.45 in an assay selected from the group consisting of: cell-based assays, fluorescent cell-based assays, high throughput screening assays, reporter cell-based assays, G protein-mediated cell-based assays, and calcium flux cell-based assays. In some other embodiments, the cell or cell line produces a Z' value of at least 0.5 in an assay selected from the group consisting of: cell-based assays, fluorescent cell-based assays, high throughput screening assays, reporter cell-based assays, G protein-mediated cell-based assays, and calcium flux cell-based assays. In some other embodiments, the cell or cell line produces a Z' value of at least 0.6 in an assay selected from the group consisting of: cell-based assays, fluorescent cell-based assays, high throughput screening assays, reporter cell-based assays, G protein-mediated cell-based assays, and calcium flux cell-based assays.

In certain embodiments, the cell or cell line stably expresses the bitter taste receptor in antibiotic-free medium for at least 2 weeks. In some other embodiments, the cell or cell line stably expresses the bitter taste receptor in antibiotic-free medium for at least 4 weeks. In some other embodiments, the cell or cell line stably expresses the bitter taste receptor in antibiotic-free medium for at least 6 weeks. In some other embodiments, the cell or cell line stably expresses the bitter taste receptor in antibiotic-free medium for at least 3 months. In some other embodiments, the cell or cell line stably expresses the bitter taste receptor in antibiotic-free medium for at least 6 months. In some other embodiments, the cell or cell line stably expresses the bitter taste receptor in antibiotic-free medium for at least 9 months.

In certain embodiments, the bitter taste receptor comprises SEQ ID NOS: 77-101. In some other embodiments, the bitter taste receptor comprises a sequence identical to SEQ id nos: 77-101, having an amino acid sequence of at least 95% identity. In some other embodiments, the bitter taste receptor comprises an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions to a nucleic acid sequence comprising SEQ ID NOS: 51-75, in a nucleic acid sequence complementary to the reverse of any one of the sequences. In some other embodiments, the bitter taste receptor comprises a polypeptide consisting of a sequence set forth as SEQ ID NOS: 51-75, or a variant allele thereof.

In certain embodiments, the bitter taste receptor comprises a polypeptide consisting of SEQ ID NOS: 51-75 in the sequence of any one of the nucleotide sequences encoding the amino acid sequence. In some other embodiments, the bitter taste receptor comprises a polypeptide consisting of a sequence identical to SEQ ID NOS: 51-75, or a nucleotide sequence having at least 95% identity thereto. In some other embodiments, the bitter taste receptor comprises an amino acid sequence encoded by a sequence of nucleic acids that hybridizes under stringent conditions to a nucleic acid sequence comprising SEQ ID NOS: 51-75, in a nucleic acid sequence complementary to the reverse of any one of the sequences. In some other embodiments, the bitter taste receptor comprises a polypeptide consisting of a sequence set forth as SEQ ID NOS: 51-75, or a variant allele of any one of seq id No. 51-75.

In certain embodiments, the bitter taste receptor is a functional bitter taste receptor. In certain embodiments, the cell or cell line changes in intracellular free calcium concentration when contacted with isoproterenol. In certain embodiments, isoproterenol has an EC50 value of about 1nM to about 20nM in a dose response curve performed using a cell or cell line. In certain embodiments, the cell or cell line has a signal to noise ratio greater than 1.

According to another aspect of the invention, there is provided a collection of cells or cell lines engineered to stably express a bitter taste receptor. In certain embodiments, the collection comprises 2 or more cells or cell lines, each stably expressing a different bitter taste receptor or allelic variant thereof. In certain embodiments, the collection additionally includes cells or cell lines engineered to stably express bitter taste receptors with known ligands. In certain embodiments, the allelic variant is a Single Nucleotide Polymorphism (SNP). In some other embodiments, each cell or cell line changes in intracellular free calcium concentration when contacted with isoproterenol. In certain embodiments, isoproterenol has an EC50 value of about 1nM to about 20nM in a dose-response curve performed with each cell or cell line.

In certain embodiments, the cells or cell lines are matched to share the same physiological properties, thereby allowing parallel processing. In certain embodiments, the physiological property is growth rate. In some other embodiments, the physiological property is adhesion to a tissue culture surface. In some other embodiments, the physiological property is a Z' factor. In some other embodiments, the physiological property is the expression level of a bitter taste receptor.

In some other embodiments of the invention, the collection comprises 2 or more cells or cell lines, each stably expressing the same bitter taste receptor or allelic variant thereof. In certain embodiments, the collective additionally comprises cells or cell lines engineered to stably express a bitter taste receptor with a known ligand. In certain embodiments, the allelic variant is a SNP. In some other embodiments, each cell or cell line changes in intracellular free calcium concentration when contacted with isoproterenol. In certain embodiments, isoproterenol has an EC50 value of about 1nM to about 20nM in a dose-response curve performed with each cell or cell line.

In certain embodiments, the cells or cell lines are matched to share all of the same physiological properties, thereby allowing parallel processing. In certain embodiments, the physiological property is growth rate. In some other embodiments, the physiological property is adhesion to a tissue culture surface. In some other embodiments, the physiological property is a Z' factor. In some other embodiments, the physiological property is the expression level of a bitter taste receptor.

According to another aspect of the invention, a method of producing a cell stably expressing a bitter taste receptor is provided. The method comprises the following steps: a) introducing a nucleic acid encoding a bitter taste receptor into a plurality of cells; b) introducing a molecular beacon that detects expression of a bitter taste receptor into the plurality of cells provided in step (a); and c) isolating cells expressing a bitter taste receptor. In certain embodiments, the method further comprises the step of generating a cell line from the cells isolated in step (c). In certain embodiments, the resulting cell line stably expresses the bitter taste receptor in media without any antibiotic selection for at least 2 weeks. In some other embodiments, the resulting cell line stably expresses the bitter taste receptor in a medium without any antibiotic selection for at least 4 weeks. In some other embodiments, the resulting cell line stably expresses the bitter taste receptor in media without any antibiotic selection for at least 6 weeks. In some other embodiments, the resulting cell line stably expresses the bitter taste receptor in a medium without any antibiotic selection for at least 3 months. In some other embodiments, the resulting cell line stably expresses the bitter taste receptor in a medium without any antibiotic selection for at least 6 months. In some other embodiments, the resulting cell line stably expresses the bitter taste receptor in a medium without any antibiotic selection for at least 9 months.

In certain embodiments, the cell used to produce the cell stably expressing the bitter taste receptor is a eukaryotic cell. In some other embodiments, the cell used to produce the cell stably expressing the bitter taste receptor is a mammalian cell. In some other embodiments, the cells used to produce cells stably expressing a bitter taste receptor do not express an endogenous bitter taste receptor. In some other embodiments, the cell used to produce the cell stably expressing the bitter taste receptor is a eukaryotic cell that does not express a cell type of endogenous bitter taste receptor. In some other embodiments, the cell used to produce a cell that stably expresses a bitter taste receptor is a mammalian cell of a cell type that does not express an endogenous bitter taste receptor. In certain embodiments, the cell used to generate a cell stably expressing a bitter taste receptor is a human embryonic kidney 293T cell.

In certain embodiments, the bitter taste receptor is a mammalian bitter taste receptor. In some other embodiments, the bitter taste receptor is a human bitter taste receptor. In some other embodiments, the bitter taste receptor does not have a polypeptide tag at its amino-terminus or carboxy-terminus. In some other embodiments, the bitter taste receptor is a mammalian bitter taste receptor that does not have a polypeptide tag at its amino-terminus or carboxy-terminus. In some other embodiments, the bitter taste receptor is a human bitter taste receptor that does not have a polypeptide tag at its amino-terminus or carboxy-terminus.

In certain embodiments, the bitter taste receptor comprises SEQ ID NOS: 77-101. In some other embodiments, the bitter taste receptor comprises a sequence identical to SEQ id nos: 77-101, having an amino acid sequence of at least 95% identity. In some other embodiments, the bitter taste receptor comprises a polypeptide encoded by a sequence that hybridizes under stringent conditions to a sequence comprising SEQ ID NOS: 51-75, or a nucleic acid that hybridizes to the nucleic acid of the reverse complement of any one of 51-75. In some other embodiments, the bitter taste receptor comprises a polypeptide encoded by a sequence that is seq id NOS: 51-75, or a variant allele thereof.

In certain embodiments, the bitter taste receptor comprises a polypeptide consisting of SEQ ID NOS: 51-75 in the sequence of any one of the nucleotide sequences encoding the amino acid sequence. In some other embodiments, the bitter taste receptor comprises a polypeptide consisting of a sequence identical to SEQ ID NOS: 51-75, or a nucleotide sequence having at least 95% identity thereto. In some other embodiments, the bitter taste receptor comprises a polypeptide encoded by a sequence that hybridizes under stringent conditions to a sequence comprising SEQ ID NOS: 51-75, or a reverse complement of any one of 51-75, or a sequence encoding a nucleic acid that hybridizes to the nucleic acid. In some other embodiments, the bitter taste receptor comprises a polypeptide consisting of a sequence set forth as SEQ ID NOS: 51-75, or a variant allele of any one of seq id No. 51-75.

In certain embodiments, the bitter taste receptor is a functional bitter taste receptor. In certain embodiments, the cells isolated in step (c) have a change in intracellular free calcium concentration when contacted with isoproterenol. In certain embodiments, isoproterenol has an EC50 value of about 1nM to about 20nM in a dose response curve performed with the cells.

In certain embodiments, the separation utilizes a fluorescence activated cell sorter.

In certain embodiments, the cells used to produce cells stably expressing a bitter taste receptor stably express endogenous G protein. In some other embodiments, the cells used to produce cells stably expressing a bitter taste receptor stably express a heterologous G protein. In some other embodiments of the invention, the cells used to produce cells stably expressing a bitter taste receptor express stably expressed endogenous and heterologous G proteins.

In certain embodiments, the method of generating a cell line that expresses a bitter taste receptor further comprises introducing a nucleic acid encoding a G protein into the cell. In certain embodiments, the nucleic acid encoding a G protein is introduced into the cell prior to introducing the nucleic acid encoding a bitter taste receptor. In some other embodiments, the nucleic acid encoding a G protein is introduced into the cell after the nucleic acid encoding a bitter taste receptor is introduced. In some other embodiments, the nucleic acid encoding the G protein is introduced at the same time as the nucleic acid encoding the bitter taste receptor. In certain embodiments, the nucleic acid encoding a bitter taste receptor and the nucleic acid encoding a G protein are on a single vector. In certain embodiments, the method further comprises introducing a molecular beacon that detects expression of a G protein into the cell prior to introducing the molecular beacon that detects expression of a bitter taste receptor. In some other embodiments, the method further comprises introducing a molecular beacon that detects expression of a G protein into the cell after introducing the molecular beacon that detects expression of a bitter taste receptor. In some other embodiments, the method further comprises introducing into the cell simultaneously a molecular beacon that detects expression of a bitter taste receptor and a molecular beacon that detects expression of a G protein. In certain embodiments, the molecular beacon that detects expression of a bitter taste receptor and the molecular beacon that detects expression of a G protein are different molecular beacons. In some other embodiments, the molecular beacon that detects expression of a bitter taste receptor and the molecular beacon that detects expression of a G protein are the same molecular beacon. In certain embodiments, the method further comprises isolating the cell expressing the G protein prior to isolating the cell expressing the bitter taste receptor, thereby isolating the cell expressing the bitter taste receptor and the G protein. In some other embodiments, the method further comprises isolating the cell expressing the G protein after isolating the cell expressing the bitter taste receptor, thereby isolating the cell expressing the bitter taste receptor and the G protein. In some other embodiments, the method further comprises simultaneously isolating the cell expressing the bitter taste receptor and the cell expressing the G protein, thereby isolating the cell expressing the bitter taste receptor and the G protein.

According to another aspect of the invention, a method of identifying a modulator of bitter taste receptor function comprises: a) contacting a cell or cell line stably expressing a bitter taste receptor with a test compound; and b) detecting a change in the function of the bitter taste receptor. In certain embodiments, the detection utilizes an assay that measures intracellular free calcium. In certain embodiments, intracellular free calcium is measured using one or more calcium-sensitive fluorescent dyes, a fluorescence microscope, and optionally a fluorescent plate reader, wherein at least one fluorescent dye binds to free calcium. In some other embodiments, intracellular free calcium is monitored by real-time imaging using one or more calcium-sensitive fluorescent dyes, wherein at least one fluorescent dye binds to free calcium.

In certain embodiments, the cell or cells in the cell line are eukaryotic cells. In some other embodiments, the cell or cells in the cell line are mammalian cells. In some other embodiments, the cell or cells in the cell line do not express an endogenous bitter taste receptor. In some other embodiments, the cell or cells in the cell line are eukaryotic cells of a cell type that does not express an endogenous bitter taste receptor. In some other embodiments, the cell or cells in the cell line are mammalian cells of a cell type that does not express an endogenous bitter taste receptor. In certain embodiments, the cell or cells in the cell line are human embryonic kidney 293T cells.

In certain embodiments, the bitter taste receptor comprises SEQ ID NOS: 77-101. In some other embodiments, the bitter taste receptor comprises a sequence identical to SEQ id nos: 77-101, having an amino acid sequence of at least 95% identity. In some other embodiments, the bitter taste receptor comprises a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide comprising SEQ ID NOS: 51-75, or a nucleic acid that hybridizes to the nucleic acid of the reverse complement of any one of 51-75. In some other embodiments, the bitter taste receptor comprises a polypeptide encoded by a sequence that is seq id NOS: 51-75, or a variant allele thereof.

In certain embodiments, the bitter taste receptor comprises a polypeptide consisting of SEQ ID NOS: 51-75 in the sequence of any one of the nucleotide sequences encoding the amino acid sequence. In some other embodiments, the bitter taste receptor comprises a polypeptide consisting of a sequence identical to SEQ ID NOS: 51-75, or a nucleotide sequence having at least 95% identity thereto. In some other embodiments, the bitter taste receptor comprises a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide comprising SEQ ID NOS: 51-75, or a reverse complement of any one of 51-75, or a sequence encoding a nucleic acid that hybridizes to the nucleic acid. In some other embodiments, the bitter taste receptor comprises a polypeptide consisting of a sequence set forth as SEQ ID NOS: 51-75, or a variant allele of any one of seq id No. 51-75.

In certain embodiments, the concentration of free calcium within the cell or cell line is altered. In certain embodiments, isoproterenol has an EC50 value of 1nM to 20nM in a dose-response curve performed using a cell or cell line.

In certain embodiments, the test compound is a bitter taste receptor inhibitor. In certain embodiments, the method further comprises contacting the cell or cell line with a known agonist of a bitter taste receptor prior to contacting the cell or cell line with the test compound. In some other embodiments, the method further comprises contacting the cell or cell line with a known agonist of a bitter taste receptor at the same time as the step of contacting the cell or cell line with the test compound.

In certain embodiments, the test compound is a bitter taste receptor agonist. In certain embodiments, the method further comprises contacting the cell or cell line with a known inhibitor of a bitter taste receptor prior to contacting the cell or cell line with the test compound. In some other embodiments, the method further comprises contacting the cell or cell line with a known inhibitor of a bitter taste receptor at the same time as the step of contacting the cell or cell line with the test compound.

In certain embodiments, the test compound is a small molecule. In certain embodiments, the test compound is a chemical moiety. In certain embodiments, the test compound is a polypeptide. In certain embodiments, the test compound is an antibody. In certain embodiments, the test compound is a food extract.

In a further aspect of the invention, a method of identifying a modulator of bitter taste receptor function comprises a) contacting a collection of cell lines with a library of different test compounds, wherein the collection of cell lines comprises 2 or more cell lines, each cell line stably expressing the same bitter taste receptor or allelic variant thereof, and wherein each cell line is contacted with one or more test compounds in the library; and b) detecting a change in function of the bitter taste receptor or allelic variant thereof stably expressed by each cell line. In certain embodiments, the detection utilizes an assay that measures intracellular free calcium. In certain embodiments, intracellular free calcium is measured using one or more calcium-sensitive fluorescent dyes, a fluorescence microscope, and optionally a fluorescent plate reader, wherein at least one fluorescent dye binds to free calcium. In some other embodiments, intracellular free calcium is monitored by real-time imaging using one or more calcium-sensitive fluorescent dyes, wherein at least one fluorescent dye binds to free calcium.

In certain embodiments, the library is a small molecule library. In certain embodiments, the library is a combinatorial library. In certain embodiments, the library is a peptide library. In certain embodiments, the library is an antibody library.

In certain embodiments, the method further comprises contacting the collection of cell lines with a known bitter taste receptor agonist prior to or simultaneously with step (a). In some other embodiments, the method further comprises contacting the collection of cell lines with a known bitter taste receptor inhibitor prior to or simultaneously with step (a).

According to another aspect of the invention, a method of identifying a modulator of bitter taste receptor function comprises: a) contacting a collection of cell lines with a test compound, wherein the collection of cell lines comprises 2 or more cell lines, each cell line stably expressing a different bitter taste receptor or allelic variant thereof; and b) detecting a change in the function of the bitter taste receptor stably expressed by each cell line. In certain embodiments, the detection utilizes an assay that measures intracellular free calcium. In certain embodiments, intracellular free calcium is measured using one or more calcium-sensitive fluorescent dyes, a fluorescence microscope, and optionally a fluorescent plate reader, wherein at least one fluorescent dye binds to free calcium. In some other embodiments, intracellular free calcium is monitored by real-time imaging using one or more calcium-sensitive fluorescent dyes, wherein at least one fluorescent dye binds to free calcium.

In certain embodiments, the test compound is a small molecule in certain embodiments. In certain embodiments, the test compound is a chemical moiety. In certain embodiments, the test compound is a polypeptide. In certain embodiments, the test compound is an antibody. In certain embodiments, the test compound is a food extract.

According to another aspect of the invention, cells engineered to stably express bitter taste receptors at a consistent level over time are produced by a method comprising the steps of: a) providing a plurality of cells expressing mRNA encoding a bitter taste receptor; b) individually dispersing cells into a single culture vessel, thereby providing a plurality of isolated cell cultures; c) culturing the cells under a desired set of culture conditions using an automated cell culture process characterized by substantially identical conditions for each isolated cell culture, normalizing the number of cells in each isolated cell culture during the culturing process, and wherein the isolated cultures are passaged according to the same protocol; d) assaying the isolated cell culture to measure expression of the bitter taste receptor at least 2 times; and e) identifying an isolated cell culture that expresses a bitter taste receptor at a consistent level in both assays, thereby obtaining the cell.

In certain embodiments, cells endogenously expressing a bitter taste receptor can be isolated from a population of cells as described herein. Such isolated cells can be used with the methods and compositions described herein (e.g., screening methods and groups of subjects).

In certain embodiments, the present invention provides a method for determining the chemical space of a compound that modulates a protein complex, wherein the method comprises:

a) contacting a plurality of chemically distinct compounds with cells that have been engineered to express a protein complex, respectively;

b) determining the effect of the compound on the activity of the protein complex;

c) correlating the effect obtained in step b) with the structural commonality of the compounds.

In certain embodiments, the present invention provides methods for identifying structural commonalities between compounds that modulate protein complexes, wherein the methods comprise:

(a) identifying a compound that modulates a protein complex according to steps a) and b) of the above method;

(b) constructing a structure-activity relationship (SAR) model for each of said compounds using a sub-molecular library for each compound and an activity profile for each compound, wherein said molecular representation for each of said compounds comprises a structure descriptor (descriptor) for each compound, wherein each of said activity profiles comprises a quantitative measure of the effect of each compound on the biological activity of the protein complex, and wherein each of said SAR models relates a structural characteristic of each compound to the activity profile of each compound;

(c) Identifying one or more structural features of each of the compounds that correlate with an activity profile of each compound based on the SAR model for each compound; and

(d) identifying at least one structural feature common to said compounds from the one or more structural features of each of said compounds identified in step (c).

In certain embodiments, the present invention provides methods for identifying a structural commonality in a compound that modulates a protein complex, wherein the method comprises:

(a) separately contacting a plurality of candidate compounds with cells that have been engineered to express the protein complex;

(b) determining the effect of each candidate compound of said plurality of compounds on the activity of said protein complex to provide an activity profile for each said candidate compound, wherein each said activity profile comprises a quantitative measure of the effect of each candidate compound on the biological activity of the protein complex;

(c) identifying one or more candidate compounds that modulate the activity of the protein complex based on the activity profile of the candidate compound;

(d) constructing a structure-activity relationship (SAR) model for each of the one or more candidate compounds identified in step (c) using a molecular representation of each candidate compound and an activity profile of each candidate compound, wherein the molecular representation of each of the one or more compounds includes a structure descriptor for each candidate compound, and wherein each of the SAR models relates a structural feature of each candidate compound to the activity profile of each candidate compound;

(e) Identifying one or more structural features of each of the one or more candidate compounds that are correlated with the activity profile of each candidate compound based on the SAR model of each candidate compound; and

(f) identifying at least one structural feature common to said one or more candidate compounds from the one or more structural features of each of said candidate compounds identified in step (e).

In more specific embodiments, the molecular representation of each of the compounds further comprises physicochemical data for the respective compound, spatial data for the respective compound, topological data for the respective compound, or a combination thereof.

In a more specific embodiment, the protein complex is a bitter taste receptor.

In a more specific embodiment, said constructing said SAR model for each of said compounds comprises applying a regression method to determine a relationship between said molecular representation of each compound and said activity profile of each compound.

In more specific embodiments, the SAR model for each of the compounds is independently a receptor-dependent free-ability field QSAR (FEFF-QSAR) model, a receptor-independent three-dimensional QSAR (3D-QSAR), or a receptor-dependent or receptor-independent four-dimensional QSAR (4D-QSAR).

In certain aspects, provided herein are kits useful for the methods described herein. In particular, provided herein are kits comprising one or more cells or cell lines stably expressing one or more complex targets. In certain embodiments, the kits provided herein comprise one or more signaling probes described herein. In particular embodiments, a kit may include one or more vectors encoding one or more complex targets. In particular embodiments, the kit includes one or more dyes for cell-based functional assays (e.g., calcium flux assays, membrane potential assays) to screen and select cells that stably express one or more complex targets.

In certain aspects, provided herein are kits comprising one or more containers filled with one or more reagents and/or cells described herein, e.g., one or more cells, vectors, and/or signaling probes provided herein. Optionally such containers may be accompanied by a brief introduction to the instructions for use that include the components of the kit.

Drawings

FIGS. 1-3 are each a schematic representation of computer program modules that may be used in accordance with the present invention.

Figure 4 includes 4 graphs, each showing a dose response curve for several compounds, each of which was tested against 4 different NaV 1.7 cell lines expressing NaV α, β 1 and β 2 subunits.

Figure 5 is a table showing the response of different NaV 1.7 cell lines expressing NaV α, β 1 and β 2 subunits to different doses of different compounds grouped by boxes.

Figure 6 is a bar graph representing data obtained after an assay based on cells expressing the umami Receptor (RNA) in which the cells expressing the umami receptor were treated with 12.5mM fructose (sweet taste agonist) or 25mM msg (umami agonist) and cultured under different conditions, in which the results of the assay from aliquots of the same cells tested in 3 of such conditions (1, 2 and "final") are illustrated.

FIG. 7 is a graphical representation of a quantitative gene expression analysis of the cell lines of the present invention expressing the umami receptors T1R1 and T1R3 subunit and Ga15 protein. Total RNA was extracted from cell lines for TaqMan analysis of gene expression using gene-specific primers and probes for T1R1, T1R3, and Ga 15. Relative expression levels relative to control cells are shown.

Figure 8 shows a representative gel that analyzes the stability of umami receptor expression in cell lines after 9 months of culture. Single-terminal RT-PCR (Single end RT-PCR) was used to estimate the expression of the T1R1, T1R3 and Ga15 genes in 1 and 9 month cultures of cell lines expressing umami receptors. Responses were also performed with and without reverse transcriptase ("+ RT" or "-RT") and samples using control cells ("control") or water alone ("none"), and as a positive control, expression of the plasmid-encoded neomycin resistance gene ("neo"). Arrows indicate responses in which positive results are expected. Due to the large number of reactions, data from different gels were juxtaposed in numbers. The data for T1R3 is shown in its own chart, since these reactions require independent PCR conditions.

Figure 9 is a series of representative response traces generated after performing an assay based on cells expressing the umami receptor. Assays based on cells expressing the umami receptor were performed using buffer alone as a control (framed wells) and 33mM of the umami receptor agonist MSG in the remaining wells. Cell lines produce Z' values greater than 0.8.

Figure 10 is a line graph showing data obtained after a dose response curve experiment using the umami receptor agonist MSG in an assay based on cells expressing the umami receptor. The response of the umami receptor expressing cell line and the control cell line is plotted as a function of agonist concentration.

FIG. 11 is a series of representative response traces generated after performing an assay based on cells expressing the umami receptor in the presence of different concentrations of MSG (1mM-100mM, right to left) and the enhancer IMP (0mM-30mM, bottom to top).

Figure 12 is a series of representative reaction traces generated after performing assays based on cells expressing umami receptor in the presence of varying concentrations of sodium cyclamate.

Figure 13 is a graph showing the different functional activities (the "assay response" on the y-axis) of native (circles) and labeled (squares) human bitter taste receptors in the presence of a range of concentrations of bitter extract (x-axis).

Figure 14 is a table showing that cell lines expressing human bitter taste receptors showed a positive rate of up to 89% for functional receptor responses. Each cell represents a well in a 96-well plate. Black boxes indicate no cells present/too few cells present. White boxes indicate cells present, but no agonist signal above the background signal of the wells. The grey boxes indicate the presence of cells where the agonist signal is above the background signal of the well.

FIG. 15 is a series of fluorescence micrographs of real-time imaging of bitter taste receptor response to bitter taste agonist in (1) cell lines expressing bitter taste receptors isolated according to the methods of the present invention (top panel) and (2) cells subjected to drug selection (bottom panel).

Figure 16 is a graph showing a dose response curve for the relative response to isoproterenol in the 25 human bitter taste receptor-Ga 15 cell lines.

Figure 17 is a table showing broadly-regulated, moderately-regulated selective bitter taste receptors as identified in transient transfection assays.

Figure 18 is a table showing the activity of different compounds on 25 different human bitter taste receptors, measured in a functional cell-based assay and expressed as a percentage activity above the baseline activity of the receptor.

Figure 19 is a table showing different bitter taste receptor assignments using native cell lines (upper row) and labeled cell lines (lower row).

Figure 20 is a bar graph representing assays based on cells expressing sweet taste Receptors (RNA) performed using 12.5mM fructose (sweet taste agonist) or 25mM MSG (umami agonist) as test compounds. Cultures were grown under different conditions, and the results of the assays from aliquots of the same cells tested in 3 of these conditions (1, 2 and "final") are illustrated in the figure. Assay reactions were normalized to the values of control cells.

FIG. 21 is a graphical representation of a quantitative gene expression analysis of cell lines of the invention expressing the sweet taste receptors human T1R2 and T1R3 subunits (SEQ ID NOS: 31 and 32, respectively) and mouse Ga15 protein (SEQ ID NO: 33). Total RNA was extracted from cell lines and gene expression analysis was performed on total RNA by TaqMan using gene specific primers and probes for human T1R2 and T1R3 and mouse Ga 15. Relative expression levels above control cells are shown.

Figure 22 shows a representative gel that analyzes the stability of sweet taste receptor expression in cell lines of the present invention after 9 months of culture. Human T1R2, human T1R3 and mouse Ga15 gene expression was assessed in 1 and 9 month cultures of cell lines expressing sweet taste receptors using single endpoint RT-PCR. Responses with and without reverse transcriptase ("+ RT" or "-RT") and with control cells ("control") or with water ("no") samples alone were also performed, and as a positive control, expression of the plasmid-encoded neomycin resistance gene ("neo"). Arrows indicate lanes in which positive results are expected. Due to the large number of reactions, data from different gels were digitally collocated. Data for the human T1R3 subunit is shown in its own chart, since these reactions require independent PCR conditions.

Figure 23 is a series of representative response traces generated after performing assays based on cells expressing sweet taste receptors (using cells of the invention) in alternate wells with the known agonist fructose at 75mM sweet taste receptor, in other wells with buffer alone used as a control. Cell lines produce Z' values greater than 0.8.

Fig. 24(a-C) are a series of line graphs showing data obtained in different dose-response experiments using sweet taste receptor agonists in assays based on cells expressing sweet taste receptors (using cells of the invention). (A) The response of cell lines expressing sweet taste receptors to natural caloric sweeteners (caloric sweentener) was plotted as a function of agonist concentration. (B) Dose response curves for common artificial sweeteners in assays based on cells expressing sweet taste receptors. (C) Dose response curves based on natural high intensity sweeteners in assays expressing sweet taste receptors.

Figure 25 is a series of representative response traces generated after performing assays based on cells expressing sweet taste receptors using cells of the invention. In contrast to the typical bell-shaped GPCR response observed with most agonists, melleo agonists show a delayed response in the calcium flux FDSS assay.

FIG. 26(A-B) depicts the genomic locus of T1R 2. Fig. 26A depicts the location of the T1R2 locus within chromosome 1. FIG. 26B depicts one possible intron-exon coding structure of the T1R2 gene. Information was obtained from the genome browser of Univrerity of California (Santa Cruz). The exons and introns corresponding to T1R2 are shown numerically in the 5 'to 3' orientation. Exon numbers are shown in black and intron numbers in gray. Information was obtained from the gene browser of Univrerity of California (Santa Cruz).

FIG. 27(A-B) depicts the genomic locus of T1R 3. FIG. 27A depicts the location of the T1R3 locus within chromosome 1. Information was obtained from the genome browser of Univrerity of California (Santa Cruz). FIG. 27B depicts one possible intron-exon coding structure of the T1R3 gene. The corresponding exons and introns of T1R3 are shown numerically in the 5 'to 3' orientation. Exon numbers are shown in black and intron numbers in gray. Scale and chromosome position are shown. Information was obtained from the gene browser of Univrerity of California (Santa Cruz).

Figure 28(a-B) depicts a representative trace of functional cell-based response to fructose addition (to a final concentration of 15 mM) by cells of the invention compared to background. Cells cultured from a single isolated cell (black trace) were tested compared to control cells (grey trace). The cells showed a higher response to fructose as shown by subtracting the control response or background from the test and control cell samples. Cell-based assays are designed to report calcium flux using fluorescent calcium signaling dyes. Fluorometric reactions were plotted along the Y-axis versus time along the X-axis. The arrows indicate the time points when fructose was added. Fig. 28A depicts traces from cells cultured from a single HuTu cell compared to a control. Fig. 28B depicts traces from cells cultured from a single H716 cell compared to a control. Figure 28C depicts traces from cells cultured from a single 293T cell compared to a control.

Fig. 29(a-B) depicts representative traces of functional cell-based responses of human odorant receptor-expressing cells to Helional (Helional) and p-tert-butyl-benzenepropanal (Bourgeonal) compared to background. Figure 29A depicts a representative trace of functional cell-based response of the cell lines described herein expressing the human odorant receptor OR3a1 to helional (to a final concentration of 4.5 mM) compared to DMSO vehicle background signal as a control. Test (black) and control (grey) traces were overlaid. The cells showed a response to helional above background. Cell-based assays are designed to report calcium flux using fluorescent calcium signaling dyes. The fluorometric reactions are plotted along the Y-axis, with time along the X-axis. Arrows indicate the time points of addition of helional or DMSO.

Figure 29B depicts a representative trace of functional cell-based response of the cell lines described herein expressing the human odorant receptor OR1D2 to p-tert-butyl-phenylpropionaldehyde (to a final concentration of 0.3 mM) compared to DMSO vehicle background signal as a control. Test (black) and control (grey) traces were overlaid. The cells showed a response to p-tert-butylphenylaldehyde above background. Cell-based assays are designed to report calcium flux using fluorescent calcium signaling dyes. The fluorometric reactions are plotted along the Y-axis, with time along the X-axis. The arrows indicate the time points of addition of p-tert-butyl-phenylpropionaldehyde or DMSO.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although exemplary methods and materials are described below, methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. In case of conflict, the present specification, including definitions, will control.

All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, such citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

Throughout this specification and the claims, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. The materials, methods, and examples are illustrative only and not intended to be limiting.

In order that the invention may be more readily understood, certain terms are first defined. Additional definitions are shown throughout the detailed description.

The term "stable" or "stably expressed" is intended to distinguish the cells and cell lines of the invention from cells that transiently express a protein, as the terms "stably expressed" and "transiently expressed" will be understood by those of ordinary skill in the art. As used herein, "not expressed in cells of the same type" includes not in any other cells of the same type, or in at least 99% of cells of the same type, or in at least 98% of cells of the same type, or in at least 97% of cells of the same type, or in at least 96% of cells of the same type, or in at least 95% of cells of the same type, or in at least 94% of cells of the same type, or in at least 93% of cells of the same type, or in at least 92% of cells of the same type, or in at least 91% of cells of the same type, or in at least 90% of cells of the same type, or in at least 85% of cells of the same type, or in at least 80% of cells of the same type, or in at least 75% of cells of the same type, or in at least 70% of cells of the same type, or in at least 60% of cells of the same type, or at least 50% of the same cell type.

As used herein, a "functional" RNA or protein of interest is an RNA or protein that has a signal-to-noise ratio of greater than 1: 1 in a cell-based assay. In certain embodiments, the "functional" RNA or protein of interest has a signal to noise ratio of greater than 2. In certain embodiments, the "functional" RNA or protein of interest has a signal to noise ratio of greater than 3. In certain embodiments, the "functional" RNA or protein of interest has a signal to noise ratio of greater than 4. In certain embodiments, the "functional" RNA or protein of interest has a signal to noise ratio of greater than 5. In certain embodiments, the "functional" RNA or protein of interest has a signal to noise ratio of greater than 10. In certain embodiments, the "functional" RNA or protein of interest has a signal to noise ratio of greater than 15. In certain embodiments, the "functional" RNA or protein of interest has a signal to noise ratio of greater than 20. In certain embodiments, the "functional" RNA or protein of interest has a signal to noise ratio of greater than 30. In certain embodiments, the "functional" RNA or protein of interest has a signal to noise ratio of greater than 40. In certain embodiments, the signal-to-noise ratio does not change by more than 10%, 20%, 30%, 40%, 50%, 60%, or 70%. In certain embodiments, the signal-to-noise ratio does not vary by more than 10%, 20%, 30%, 40%, 50%, 60%, or 70% between experiments. In certain embodiments, the signal-to-noise ratio does not change by more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% between experiments. In certain embodiments, the signal-to-noise ratio does not vary by more than 10%, 20%, 30%, 40%, 50%, 60%, or 70% between 2 to 20 different replicates of an experiment. In certain embodiments, the signal-to-noise ratio does not vary by more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% between 2 to 20 different replicates of an experiment. In certain embodiments, the signal to noise ratio does not change by more than 10%, 20%, 30%, 40%, 50%, 60%, or 70% for cells tested from 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, or more than 70 days (wherein the cells are in continuous culture). In certain embodiments, the signal to noise ratio does not change by more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% for cells tested from 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, or more than 70 days (wherein the cells are in continuous culture). In certain embodiments, a functional protein or RNA of interest has one or more biological activities of a naturally occurring or endogenously expressed protein or RNA.

The term "cell line" or "clonal cell line" refers to a population of cells that are progeny of a single cell of origin. As used herein, cell lines are maintained in cell culture in vitro, and may be frozen in aliquots to establish clonal cell banks.

The term "stringent conditions" or "stringent hybridization conditions" describes temperature and salt conditions used to hybridize one or more nucleic acid probes to a nucleic acid sample and wash away probes that do not specifically bind to a target nucleic acid in the sample. Stringent conditions are known to those of ordinary skill in the art and can be found, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous and non-aqueous methods are described in the schemes and either can be used. An example of stringent hybridization conditions is hybridization in 6 XSSC at about 45 ℃ followed by at least one wash in 0.2 XSSC, 0.1% SDS at 60 ℃. Another example of stringent hybridization conditions is hybridization in 6 XSSC at about 45 ℃ followed by at least one wash in 0.2 XSSC, 0.10% SDS at 65 ℃. Stringent hybridization conditions also include hybridization in 0.5M sodium phosphate, 7% SDS at 65 ℃ followed by at least one wash in 0.2 XSSC, 1% SDS at 65 ℃.

The phrase "percent equivalent" or "percent identity" in relation to amino acid and/or nucleic acid sequences refers to similarity between at least 2 different sequences. Percent identity can be determined by standard alignment algorithms, for example, Basic Localalignment Tool (BLAST) described by Altsull et al ((1990) J.mol.biol., 215: 403-410); the algorithm of Needleman et al ((1970) J.mol.biol., 48: 444-453); or Meyers et al ((1988) Compout. appl. biosci., 4: 11-17). One set of parameters may be a Blosum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The percent identity between 2 amino acid or nucleotide sequences can also be determined using the algorithm of e.meyers and w.miller ((1989) cabaos, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. Percent identity is typically calculated by comparing sequences of similar length.

Protein analysis software matches similar amino acid sequences using similarity measures specified for various substitutions, deletions or other modifications, including conservative amino acid substitutions. For example, GCG Wisconsin Package (Accelrys, Inc.) contains programs such as "Gap" and "Bestfit" that can be used, along with default parameters, to determine sequence identity between closely related polypeptides (e.g., homologous polypeptides from different species) or between a wild-type protein and its mutant protein. See, e.g., GCG version 6.1. Polypeptide sequences can also be compared using FASTA, using default or recommended parameters. Programs FASTA in GCG version 6.1 (e.g., FASTA2 and FASTA3) provide alignments and percentage sequence identities of regions of optimal overlap between query and search sequences (Pearson, Methods enzymol.183: 63-98 (1990); Pearson, Methods mol.biol.132: 185-219 (2000)).

The length of polypeptide sequences compared for identity will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, usually at least about 28 residues, preferably more than about 35 residues. The length of the DNA sequences compared for identity will generally be at least about 48 nucleic acid residues, usually at least about 60 nucleic acid residues, more usually at least about 72 nucleic acid residues, generally at least about 84 nucleic acid residues, preferably more than about 105 nucleic acid residues.

The phrase "substantially as shown," "substantially identical," or "substantially homologous" in relation to an amino acid or nucleotide sequence means that the related amino acid or nucleotide sequence is identical to, or has insignificant differences from, the comparative sequence (e.g., conservative amino acid substitutions or nucleic acids encoding such substitutions). No significant differences include minor amino acid changes, such as 1 or 2 substitutions in the 50 amino acid sequence of a specified region or in the nucleic acid encoding such a sequence.

Modulators include any substance or compound that alters the activity of a protein of interest, such as a taste receptor (e.g., a bitter taste receptor, umami receptor, or sweet taste receptor). Modulators may be agonists (enhancers or activators) or antagonists (inhibitors or blockers), including partial agonists or antagonists, selective agonists or antagonists, and inverse agonists, and may also be allosteric modulators. A substance or compound is a modulator even if its modulating activity is altered under different conditions or concentrations or for different forms of the protein of interest, such as taste receptors (e.g., bitter taste receptors, umami receptors, or sweet taste receptors). In other aspects, a modulator can alter the ability of another modulator to affect the function of a protein of interest, such as a taste receptor (e.g., a bitter taste receptor, umami receptor, or sweet taste receptor). In particular embodiments, the modulators may positively or negatively alter the structure, conformation, biochemical or biophysical properties or functionality of taste receptors (e.g., bitter taste receptors, umami receptors or sweet taste receptors).

The terms "enhancer", "agonist" or "activator" are compounds or substances that increase one or more activities of a protein of interest, such as a taste receptor (e.g., a bitter taste receptor, umami receptor, or sweet taste receptor). In particular embodiments, the terms "enhancer", "agonist" or "activator" in the context of a taste receptor refer to a compound or substance that increases a downstream signaling response associated with a taste receptor (e.g., a bitter taste receptor, umami receptor, or sweet taste receptor). In particular embodiments, increasing taste receptor activity can result in a change in the amount or distribution of intracellular molecules or the activity of enzymes that are part of the intracellular signaling pathway of the bitter taste receptor. Examples of intracellular molecules include, but are not limited to, free calcium, cyclic adenosine monophosphate (cAMP), inositol monophosphate, inositol diphosphate, or inositol triphosphate. Examples of enzymes include, but are not limited to, adenylate cyclase, phospholipase C, G protein-coupled receptor kinases.

The terms "inhibitor," "antagonist," or "blocker" refer to a compound or substance that reduces or blocks one or more activities of a protein of interest, such as a taste receptor (e.g., a bitter taste receptor, umami receptor, or sweet taste receptor). In particular embodiments, the terms "inhibitor," "antagonist," or "blocker" in the context of a taste receptor refer to a compound or substance that attenuates a downstream signaling response associated with a taste receptor (e.g., a bitter taste receptor, umami receptor, or sweet taste receptor). In particular embodiments, decreasing taste receptor activity can result in a change in the amount or distribution of intracellular molecules or a change in the activity of an enzyme that is part of the intracellular signaling pathway of a bitter taste receptor. Examples of intracellular molecules include, but are not limited to, free calcium, cyclic adenosine monophosphate (cAMP), inositol monophosphate, inositol diphosphate, or inositol triphosphate. Examples of enzymes include, but are not limited to, adenylate cyclase, phospholipase C, G protein-coupled receptor kinases.

Sweet taste receptors are proteins present in many mammalian tissues, including epithelial cells of the mouth, lungs, and intestines. Without being bound by theory, it is believed that sweet taste receptor dysregulation or dysfunction may be associated with a number of disease states, including diabetes and obesity.

The phrase "functional sweet taste receptor" refers to a sweet taste receptor that comprises at least T1R2 and T1R3 subunits and that responds to known activators such as fructose, glucose, sucrose, monellin/miraculin, mogroside (mogroside), steviva, rebaudioside a (rebaudioside a), saccharin, aspartame, sodium cyclamate, sucralose, sorbitol, acesulfame K, or Gymnema Sylvestre or known inhibitors such as 4, 6-dichloro-4, 6-dideoxy-alpha-D-lactopyranoside methyl ester (MAD-di-ci-Gal) in a manner similar to (i.e., at least 50%, 60%, 70%, 80%, 90%, and 95% identical) to a sweet taste receptor produced in a cell that normally expresses the receptor without genetic modification of the cell to produce the receptor) PNP/lactitol (lactisole) which may act differentially at different concentrations, gymnemic acid 1, North Hovenin (hoduloside), ziziphin (ziziphin), and Gymnetin (gurarin). Sweet taste receptor behavior can be measured, for example, by physiological activity or pharmacological response. Physiological activities include, but are not limited to, activation of G proteins and related downstream signaling. Pharmacological responses include, but are not limited to, inhibition, activation, and modulation of a receptor. Can be used for example in the monitoring of _αqSuch responses are measured in assays of intracellular calcium release from the endoplasmic reticulum upon activation of the protein by activated sweet taste receptors.

Umami receptors are proteins present in many mammalian tissues, including epithelial cells of the mouth, lung and intestine. Without being bound by theory, it is believed that umami receptor dysregulation or dysfunction may be associated with a number of disease states, including diabetes and obesity.

The phrase "functional umami receptor" refers to an umami receptor that comprises at least T1R1 and T1R3 subunits and that responds in a similar manner (i.e., at least 50%, 60%, 70%, 80%, 90%, and 95% identical) to an umami receptor produced in a cell that normally expresses the receptor without the need for genetic engineering of the cell to produce the receptor) to a known activator such as monosodium glutamate (MSG) or a known inhibitor such as lactitol (which is known to act as an umami receptor inhibitor at a particular concentration) or PMP (2- (4-methoxyphenoxy) -propionic acid). Umami receptor behavior can be measured, for example, by physiological activity or pharmacological response. Physiological activities include, but are not limited to, activation of G proteins and related downstream signaling. Pharmacological responses include, but are not limited to, inhibition, activation, and modulation of a receptor. Can be used for example in the monitoring of _αqSuch responses are measured in assays of intracellular calcium release from the endoplasmic reticulum upon activation of the protein by an umami receptor.

The phrase "functional bitter taste receptor" refers to a bitter taste receptor that responds to a known activator or known inhibitor in a manner substantially similar to a bitter taste receptor in a cell that normally expresses the bitter taste receptor without modification. Bitter taste receptor behavior can be measured, for example, by physiological activity or pharmacological response. Physiological activity includes, but is not limited to, the perception of bitter taste. Pharmacological responses include, but are not limited to, a change in the amount or distribution of an intracellular molecule or a change in the activity of an enzyme that is part of the intracellular signaling pathway of a bitter taste receptor when the bitter taste receptor is contacted with a modulator. For example, the pharmacological response may include an increase in intracellular free calcium when the bitter taste receptor is activated, or a decrease in intracellular free calcium when the bitter taste receptor is blocked.

The term "bitter taste receptor" as used herein refers to any of the G protein-coupled receptors that are expressed on the surface of taste receptor cells and mediate the perception of bitter taste through a second messenger pathway.

By "heterologous" or "introduced" a protein of interest, such as a taste receptor subunit (e.g., a bitter taste receptor subunit, an umami receptor subunit, or a sweet taste receptor subunit) or a G protein, is meant that the protein of interest, such as a taste receptor subunit (e.g., a bitter taste receptor subunit, an umami receptor subunit, or a sweet taste receptor subunit) or a G protein, is encoded by a nucleic acid that is introduced into a host cell.

By "gene-activated" a protein of interest, such as a taste receptor subunit (e.g., a bitter taste receptor subunit, an umami taste receptor subunit, or a sweet taste receptor subunit) or a G protein, is meant that the endogenous nucleic acid encoding the subunit or protein is activated for expression by the introduction of an expression control sequence and operative linkage to the nucleic acid.

The present invention provides for the first time novel cells and cell lines produced by cells meeting the urgent need for cells stably expressing functional RNAs of interest or functional proteins of interest, including complex proteins such as heteromultimeric proteins and proteins for which the ligands are unknown. The cells and cell lines of the invention are suitable for any use where consistent functional expression of an RNA or protein of interest is desired. Applicants have generated cell lines consistent with this description for a variety of proteins, single subunits and heteromultimers (including heterodimers and proteins with more than 2 different subunits), including membrane proteins, cytosolic proteins, and secretory proteins, as well as various combinations of these proteins.

Examples of proteins of interest include, but are not limited to: receptors such as, (e.g., cytokine receptors, immunoglobulin receptor family members, ligand-gated ion channels, protein kinase receptors, G protein-coupled receptors (GPCRs), nuclear hormone receptors, and other receptors), signaling molecules (e.g., cytokines, growth factors, peptide hormones, chemokines, membrane-bound signaling molecules, and other signaling molecules), kinases (e.g., amino acid kinases, carbohydrate kinases, nucleotide kinases, protein kinases, and other kinases), phosphatases (e.g., carbohydrate phosphatases, nucleotide phosphatases, protein phosphatases, and other phosphatases), proteases (e.g., aspartic proteases, cysteine proteases, metalloproteinases, serine proteases, and other proteases), regulatory molecules (e.g., g., G protein regulators, large G proteins, proteins, Small gtpase, kinase modulators, phosphatase modulators, protease inhibitors, and other enzyme modulators), calcium binding proteins (e.g., annexins, calmodulin-related proteins, and other selective calcium binding proteins), transcription factors (e.g., nuclear hormone receptors, basal transcription factors, basic helix-loop-helix transcription factors, creb transcription factors, hmg-box transcription factors, homeobox transcription factors, other transcription factors, transcription cofactors, and zinc finger transcription factors), nucleic acid binding proteins (e.g., helicases, DNA ligases, DNA methyltransferases, RNA methyltransferases, double-stranded DNA binding proteins, restriction enzymes, replication origin binding proteins, reverse transcriptases, ribonucleoproteins, ribosomal proteins, single-stranded DNA binding proteins, centromeric DNA binding proteins, chromatin/chromatin-chromatin binding proteins, chromatin-protein fragments, protein fragments, DNA glycosylase, DNA photorepair enzyme, DNA polymerase processivity factor, DNA strand-pairing protein (DNA strand-pairing protein), DNA topoisomerase, DNA-directed DNA polymerase, DNA-directed RNA polymerase, damaged DNA-binding protein (damaged DNA-binding protein), histone, primase, endoribonuclease, deoxyriboexonuclease, exoribonuclease, translation elongation factor, translation initiation factor, translation release factor, mRNA polyadenylation factor, mRNA splicing factor, other DNA-binding proteins, other RNA-binding proteins, and other nucleic acid-binding proteins), ion channel (e.g., anion channel, ligand-gated ion channel, voltage-gated ion channel, and other ion channel), transporter (e.g., cation transporter, ATP-binding cassette (ABC) transporter, amino acid transporter, DNA polymerase, DNA topoisomerase, DNA-binding protein, and DNA polymerase, translation initiation factor, translation release factor, mRNA polyadenylation factor, mRNA splicing factor, other DNA, Carbohydrate transporters and other transporters), transfer/carrier proteins (e.g., apolipoproteins, mitochondrial carrier proteins, and other transfer/carrier proteins), cell adhesion molecules (e.g., cam family attachment molecules, cadherins, and other cell adhesion molecules), cytoskeletal proteins (e.g., actin and actin-related proteins, actin-binding motor proteins, non-motor actin-binding proteins, other actin family cytoskeletal proteins, intermediate fibers, tubulaceae cytoskeletal proteins, and other cytoskeletal proteins), extracellular matrices (e.g., extracellular matrix glycoproteins, extracellular matrix connexins, extracellular matrix structural proteins, and other extracellular matrices), cell-connecting proteins (e.g., gap connexins, tight connexins, and other cell-connecting proteins), and the like, Synthases, synthetases, oxidoreductases (e.g., dehydrogenases, hydroxylases, oxidases, oxygenases, peroxidases, reductases, and other oxidoreductases), transferases (e.g., methyltransferases, acetyltransferases, acyltransferases, glycosyltransferases, nucleotidyl transferases, phosphorylases, transaldolases, transaminases, transketolases, and other transferases), hydrolases (e.g., deacetylases, deaminases, esterases, galactosidases, glucosidases, glycosidases, lipases, phosphodiesterases, pyrophosphatases, amylases, and other hydrolases), lyases (e.g., adenylate cyclases, guanylate cyclases, aldolases, decarboxylases, dehydratases, hydratases, and other lyases), isomerases (e.g., epimerases/racemases, mutases, and other isomerases), Ligases (e.g., DNA ligases, ubiquitin protein ligases, and other ligases), defense/immune proteins (e.g., anti-microbial response proteins, complement components, immunoglobulins, immunoglobulin receptor family members, major histocompatibility complex antigens, and other defense and immune proteins), membrane trafficking proteins (e.g., membrane trafficking regulatory proteins, SNARE proteins, vesicle coat proteins, and other membrane trafficking proteins), chaperones (e.g., chaperones, hsp 70 family chaperones, hsp 90 family chaperones, and other chaperones), viral proteins (e.g., viral coat proteins, and other viral proteins), myelin proteins, other accessory function proteins (miscellaneous function proteins), storage proteins, structural proteins, surfactants, and transmembrane receptor modulator/linker proteins. Other examples of proteins and their function include those described in Paul d.thomas, Michael j.campbell, Anish Kejariwal, Huaiyu Mi, brian karlak, Robin Daverman, Karen Diemer, anusia murugaunujan, apurvanarechia.2003. panther: a library of proteins and expressed by function, genome Res., 13: 2129-2141 (which is hereby incorporated by reference) and its functions.

Examples of GPCRs include, but are not limited to:

class a GPCRs, including but not limited to: 5-hydroxytryptamine receptors (e.g., HTR1A, HTR1B, HTR1D, HTR1E, HTR1F, HTR2A, HTR2B, HTR2C, HTR4, HTR5A, HTR6, and HTR7), muscarinic acetylcholine receptors (e.g., CHRM 7, and CH3672), adenosine receptors (e.g., ADORA 7, ADORA 27, and ADORA 7), alpha-adrenoreceptors (e.g., ADRA 17, ADRA 27, and ADRA2 7), beta-adrenoreceptors (e.g., ADRB 7, and KRRB 7), anaphylatoxin receptors (e.g., ADRA1, GPR5, ADRA 27, and ADRA2 7), beta-adrenoreceptors (e.g., ADRB 7, e.g., GPR7, GPR, GRC, and GPR 7), chemokine receptors (e.g., AGPR), e.g., GRC, AGR 7, AGPR), angiotensin 7, AGC, and GPR7, e.g., GPPR 7, and GPR7, e.g., GPCR 7, e.g., angiotensin receptor (e.g., GPCR 7, and GPR7, CXCR3, CXCR4, CXCR5, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CR 9, XCR 9 and CXCR 9), cholecystokinin receptors (e.g., CCKAR and CCKBR), dopamine receptors (e.g., DRD 9 and DRD 9), endothelin receptors (e.g., EDNRA and EDNRB), estrogen receptors (e.g., GPER), formyl peptide receptors (e.g., FPR 9 and FPR 9), free fatty acid receptors (e.g., FFAR 9 and GPR 9), galanin receptors (e.g., ALR 9, GALRR 9 and GALRR 9), lyso receptor (e.g., GHHR 9, CGHR, LHR 9 and RGR 9), RGHRGNHR receptor (e.g., HRKR 9), RGR 9, HRGNHR receptor, HRGNHR 9, HRT 9 and HRT 9, HRT 9, and HRT 9, such as HRT 9, LPAR1, LPAR2, LPAR3, S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5), melanin-concentrating hormone receptors (e.g., MCHR1 and MCHR2), melanocortin receptors (e.g., MC1R, MC2R, MC3R, MC4R and MC5R), melatonin receptors (e.g., MTNR1A and MTNR1B), motilin receptors (e.g., MLNR), neurointerleukin U receptors (e.g., NMUR1 and NMUR2), neuropeptide FF/neuropeptide AF receptors (e.g., NPFFR1 and NPFFR2), neuropeptide S receptors (e.g., NPSR1), neuropeptide W/OPneuropeptide B receptors (e.g., NPBWR1 and NPBWR 1), neuropeptide Y receptors (e.g., NPNPBWR 1 and NPSR1), neuropeptide WOPOPB receptors (e.g., NPRK 1 and NPRK 1), neuropeptide WTRK receptor (e.g., NPRK 1), neuropeptide WK 1, NPRK receptor (e.g., NPRK 1), receptor family 1, NPRK 1, and NPRK 1), receptor (e.g., NPRK 1), receptor family 1, HCRTR and HCRTR), P2 receptors (e.g., P2RY, and P2 RY), peptide P518 receptors (e.g., QRFPR), platelet activin receptors (e.g., PTAFR), Prokineticin receptors (e.g., PROKR and PROKR), prolactin-releasing peptide receptors (e.g., PRLHR), prostaglandin receptors (e.g., PTGDR, PTGER, PTGFR, PTGIR, PTXA 2, and GPR), protease-activating receptors (e.g., thrombin (F2)), protease-activating receptors (e.g., F2RL, and F2 RL), relaxin family peptide receptors (e.g., RXFP, RXXFP, and RXFP), somatostatin receptors (e.g., SSTR, tachykinin receptors (e.g., SSTR, TRHR), thyroid stimulating hormone-releasing receptors (e.g., TRCR), thyroid stimulating hormone receptors (e.g., TACR), thyroid stimulating hormone receptors (TRCR), prohormone receptor (TROCR, TRCR), prohormone receptor (TROCR), Prokinetin receptor (CRR), Prokinetin, Vasopressin and oxytocin receptors (e.g., AVPR1, AVPR1, and OXTR), class A orphan receptors (e.g., GPR182, CCRL, CMKLR, CMKOR, GPR183, GPR37L, GPR101, GPR120, GPR132, GPR135, GPR139, 141 GPR, GPR142, GPRY 146, GPR148, GPR149, GPR150, GPR151, GPR152, GPR153, GPR160, LGGPR 161, GPR171, GPR173, GPR162, TAR, GPR;

Class B GPCRs, including but not limited to: calcium-sensing receptors (Calcium-sensing receptors) (e.g., CASR and GPRC6A), GABA-B receptors (e.g., GABBR1 and GABBR2), GPRC5 receptors (e.g., GPRC5A, GPRC5B, GPRC5C, and GPRC5D), metabotropic glutamate receptors (e.g., GRM1, GRM2, GRM3, GRM4, GRM5, GRM6, GRM7, and GRM8), class C orphan receptors (e.g., GPR156, GPR158, GPR179, GPRC5A, GPRC5B, GPRC5C, and GPRC 5D);

class C GPCRs, including but not limited to: calcitonin receptors (e.g., CALCR/CT, AMY1, CALCRL, CGRP, AM1, and AM 1), corticotropin releasing factor receptors (e.g., CRHR1 and CRHR 1), glucagon receptor families such as GCGR, GLP 11, GLP2 1, GIPR, SCTR, and GHRHR), parathyroid hormone receptors (e.g., PTH 11 and PTHR 1), VIP and PACAP receptors (e.g., ADCYAP1R1, VIPR1, and VIPR 1), orphan B receptors (e.g., Bal1, CD 1, CELSR1, ELTD1, EMR1, GPR110, GPR111, GPR112, hn 113, GPR115, GPR123, GPR124, GPR143, GPR1, GPR143, GPR124, GPR143, GPR1, GPR143, GPR 33, GPR1, GPR 33, GPR143, GPR;

Class D GPCRs, including but not limited to fungal mating pheromone receptors (e.g., STE2 and STE 3);

class E GPCRs, including but not limited to cAMP receptors (e.g., Dictyostelium);

class F GPCRs including, but not limited to frizzled receptors (e.g., FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, and SMO);

and

unclassified GPCRs (e.g., OPCML, OGFR, OGFRL1, and OPRS 1).

Examples of voltage-gated ion channels include, but are not limited to:

calcium-activated potassium channels including, but not limited to, KCNMA1, KCNN1, KCNN2, KCNN3, KCNN4, KCNT1, KCNT2, and KCNU 1;

CatSper and two-pore channels including, but not limited to, CatSper1, CatSper2, CatSper3, CatSper4, TPCN1, and TPCN 2;

cyclic nucleotide regulated channels including, but not limited to, CNGA1, CNGA2, CNGA3, CNGA4, CNGB1, CNGB3, HCN1, HCN2, HCN3, and HCN 4;

inwardly rectified potassium channels including, but not limited to, KCNJ1, KCNJ2, KCNJ12, KCNJ4, KCNJ14, KCNJ3, KCNJ6, KCNJ9, KCNJ5, KCNJ10, KCNJ15, KCNJ16, KCNJ8, KCNJ11, and KCNJ 13;

transient receptor potential channels including, but not limited to, TRPA1, TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6, TRPC7, TRPM1, TRPM2, TRPM3, TRPM4, TRPM5, TRPM6, TRPM7, TRPM8, MCOLN1, MCOLN2, MCOLN3, PKD2, PKD2L1, PKD2L2, TRPV1, TRPV2, TRVP3, TRPV4, TRPV5, and TRPV 6;

2-P potassium channels (Two-P potassium channels), including but not limited to KCNK1, KCNK2, KCNK3, KCNK4, KCNK5, KCNK6, KCNK7, KCNK9, KCNK10, KCNK12, KCNK13, KCNK15, KCNK16, KCNK17, and KCNK 18;

voltage gated calcium channels including, but not limited to, CACNA1S, CACNA1C, CACNA1D, CACNA1F, CACNA1A, CACNA1B, CACNA1E, CACNA1G, CACNA1H, and CACNAII;

voltage gated potassium channels including, but not limited to, KCNA1, KCNA2, KCNA3, KCNA4, KCNA5, KCNA6, KCNA7, KCNA10, KCNB1, KCNB2, KCNC1, KCNC2, KCNC3, KCNC4, KCND1, KCND2, KCND3, KCNF1, KCNG1, KCNG2, KCNQ2, KCNV2, KCNS2, KCNH2, and KCNH 2;

voltage gated sodium channels including, but not limited to, SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SCN10A, and SCN 11A; and

other voltage-gated ion channels include, but are not limited to, KCNE1, KCNE1L, KCNE2, KCNE3, KCNIP1, KCNIP2, KCNIP3, KCNIP4, KCNMB1, KCNMB2, KCNMB3, CNMB3L, and KCNMB 4.

Examples of ligand-gated ion channels include, but are not limited to:

5-hydroxytryptamine receptor subunits including, but not limited to, 5HT3Acapo, 5HT3Ahosa, 5HT3 amuu, 5HT3Amupu, 5HT3Aorcu, 5HT3Apatr, 5HT3Arano, 5HT3Bhosa, 5HT3Bmumu, 5HT3Borcu, 5HT3Bpatr, 5HT3Brano, 5HT3Chosa, 5HT3Cpatr, 5HT3Dhosa, 5HT3Dpatr, 5HT3Ehosa, 5HT3gaga, and 5HTmod1 cael; 5-HT receptors include: 5-HT 1A; 5-HT 1B; 5-HT 1D; 5-HT 1E; 5-HT 1F; 5-HT 2A; 5-HT 2B; 5-HT 2C; 5HT4 splice isoforms a, b, c, d, e, f, g, n; 5-HT 5A; 5-HT 6; 5-HT7 spliced forms a, b, c.

ACHa10gag, ACHa10hosa, ACHa10mu, ACHa10 patra, ACHa10patr, ACHa10rano, ACHa1anga, ACHa1apca, ACHa1 atara, ACHa1axela, ACHa1bota, ACHa1 btaar, ACHa1bxela, ACHa1cafa, ACHa1dare, ACHa1gaga, ACHa1hevi, ACHa1 hoda, ACHa1toca, ACHa1 momu, ACHa1mype, ACHa1naha, ACHa1nana, ACHa3 AChaac 3, AChaac 3 AChaac 7, AChaac 3, AChaac 7, AChaac 3, AChaac 4 AChaac 3, AChaac 4 ACha1 acar ACha7, ACha1 acar 3, ACha7, ACha1 acar ACha7, ACha1 acar 3, ACha2 ACha7, ACha2 ACha7, ACha2 ACha7, ACha7, ACha7, ACha7, ACha1 acar 3, ACha7, ACha1 acar ACha7, ACha2 ACha7, ACha1 acar 3, ACha2 ACha7, ACha2 ACha7, ACha2 ACha2 ACha2 ACha, ACha7, ACha2 ACha7, ACha1 acar 3, ACha1 acar 3, ACha7, ACha, ACha7, ACha7, ACha, ACHacr14cael, AChacr15cael, AChacr16cael, AChacr17cael, AChacr18cael, AChacr19cael, AChacr20cael, AChacr21cael, AChacr22cael, AChacr23cael, AChacr2cael, AChacr3cael, AChacr4cael, AChacr5cael, AChacr6cael, AChacr7cael, AChacr8cael, AChacr9cael, AChaco, ACHal1 aco, ACHal1 acgrid, ACHal1 cord, ACHalasr 1, ACHallucar, ACHal1, AChalase, ACHalys, ACHard 2, AChacar Hb3, AChacr3, AChacr Hb3, AChacr Hb Hgag Hg, AChacr3, AChacr Hgag Hg, AChacr Hgag Hg 2, AChacr Hgag Hg, AChacr4, AChacr Hgag Hg, AChacr Hgag Hg, AChacr Hgag Hg, AChacr Hgag Hg, AChacr Hgag Hg, AChacr Hgag Hg, AChacr Hgag Hg, AChacr Hgag Hg 2, AChacr Hg, AChacr Hgag Hg, AChacr Hg, AChac Hg, AChacr Hg, ACHg, AChacr Hgag Hg, ACHg, AChacr Hg, ACHg, ACHgag Hgac Hg Hgag Hg, ACHg, ACH, ACHr13a5_4cael, ACHronvo, ACHsaddrme, ACHsbddrme, ACHssu1osci, ACHssu2osci, ACHt01h10_1cael, ACHt01h10_2cael, ACHt01h10_3cael, ACHt01h10_5cael, ACHt01h10_6cael, ACHt01h10_7cael, ACHt05b4_1cael, ACHtar1trco, ACHunc29cael, ACHunc38cael, ACHunc63cael, ACHy44a6e _1cael, ACHy57g11c _2cael, ACHy57g11 _ c _49cael, ACHy 637 _1cael, HY 539 73b 685 2 _26cael and HY 2cael 73f 73;

GABAA receptor subunits including, but not limited to, GABa1bota, GABa1gaga, GABa1hosa, GABa1mumu, GABa1rara, GABa2bota, GABa2hosa, GABa2mumu, GABa2rara, GABa3bota, GABa3hevi, GABa3hosa, GABa3mumu, GABa3rara, GABa4bota, GABa4hosa, GABa4mumu, GABa4rara, GABa5hosa, GABa5rara, GABa6caau, GABa6hosa, GABa6mumu, GABa6rara, GABb1bota, GABb1hosa, GABb1mumu, GABb1 mub 1mumu, GABb1 p, GABb2dare, GABb2hosa, GABdhras 2 rab 3 Bb3 bca, GABdhgal 3 Bdhaab 3 gab 3 bca, GABdhaab 3 gab 3632, GABdhaab 5, GABdhaab 3 bca, GABdhaab 3 gab 3 3623 gab 3 3619 gab 3 3623, GABdhaab 3 gab 3 3623, GABdhaab 3 gab 3b 3 3623 b3 3623, GABdhaab 3 gab 3b 3 3623 b3 rab, GABf11h8_2cael, GABf47a4_1cael, GABf55d10_5cael, GABf58g6_4cael, GABggaga, GABghosa, GABgmumu, GABgrano, GABg2bota, GABg2gaga, GABg2hosa, GABg2mumu, GABg2rara, GABg3hosa, GABg3mumu, GABg3rano, GABg4gaga, GABg4hosa, GABg4mumu, GABgbr2cael, GABddrmre, GABhg1haco, GABk10d6_1cael, GABphosa, GABpmumu, GABr1 amonae, GABbhmoam 1, GABdhomam 3, GABbhbr 3 GABr3 gabbo, GABbhb 3 gabbo, GABbhsab 3 gabbo, GABbhg 2 gabbo, GABbhsab 3 gabbo, GABg3 gabbo, GAbbo, GAbbacae 3 gabbo, GAbbbrabbra, GAbbo, GAbbbrabbo 3 gabbo, GAbbbrabbo, GAbbacae 3 gabbo, GAbbbrabbo, GAbbbrabbacae 3 gabbo, GAbbo, GAbbacae 3 gabbacae 3 gabbo, GAbbo, GAbbacae, GAbb;

Glycine/histamine receptor subunits including, but not limited to, GLYa1dare, GLYa1hosa, GLYa1mumu, GLYa1rano, GLYa2dare, GLYa2hosa, GLYa2mumu, GLYa2rano, GLYa3dare, GLYa3hosa, GLYa3moam, GLYa3 muu, GLYa3rano, GLYa4adare, GLYa4bdare, GLYa4hosa, GLYa4mumu, GLYbdare, GLYbhosa, glybmum, GLYbrano, and HIScl1 dre;

ATP acceptor subunits including, but not limited to, ATPp2x1hosa, ATPp2x1rano, ATPp2x2capo, ATPp2x2hosa, ATPp2x2mumu, ATPp2x2rano, ATPp2x3hosa, ATPp2x3mumu, ATPp2x3rano, ATPp2x4bota, ATPp2x4gaga, ATPp2x4hosa, ATPp2x4mumu, ATPp2x4orcu, ATPp2x4rano, ATPp2x5bota, ATPp2x5hosa, ATPp2x5mumu, ATPp2x5rano, ATPp2x6hosa, ATPp2x6 muo, ATPp2x6 hosta, ATpp2x7 hosta 7 atxa, ATPp2x3 and ATPp2x3 moca;

glutamine receptor subunits including, but not limited to, GLU1_1arth, GLU1_2arth, GLU1_3arth, GLU1_4arth, GLU2_1arth, GLU2_2arth, GLU2_3arth, GLU2_4arth, GLU2_5arth, GLU2_6arth, GLU2_7arth, GLU2_8arth, GLU2_9 kbp, GLU3_1arth, GLU3_2arth, GLU3_3arth, GLU3_4arth, GLU3_5arth, GLU3_6arth, GLU 5 _7arth, GLUd1 hossa, GLUd1 mut, GLUnu 1, GLu2 arto, GLhond 2arth, GLu2 artar, GLu2 gla 2, GLu 2arth, GLu2 artar, GLu1, GLu2 acar, GLu2, GLucar, GLu2 acar, GLu1, GLu2 acar, GLu2, GLu1, GLu2 acar, GLu1, GLu2 acar, GLu2, GLu1, GLu2 acar, GLu2, GLu1, GLu2 u1, GLu1, GLu1, GLu2 acar, GLu1, GLu2, GLu1, GLu2 u1, GLu1, GLu2 u1, GLu2 u1, GLu2, GLu2 u1, GLu1, GLu2, GLu2 u1, GLu2 u2, GLu2, GLu2, GLu1, GLu1, GLu2 u2, GLu1, GLu2, GLu1, GLUr3caau, GLUr3coli, GLUr3gaga, GLUr3hosa, GLUr3 muu, GLUr3rano, GLUr4caau, GLUr4coli, GLUr4gaga, GLUr4hosa, GLUr4mumu, GLUr4rano, GLUr5daae, GLUr5hosa, GLUr5 muu, GLUr5rano, GLUr6hosa, GLUr6mumu, GLUr6rano, GLUr6xela, GLUr7 saho, GLUr7 muu, GLUr7rano, GLUrdrrly, GLUrlAdrme, GLUrlBlryme, GLUrllryst, Urlylyst, GLuclk 1 cll, GLuclol, GLUbechal, GLUxcalcalo, GLurcol, GLUxcalcalo 3 and GLUxcalcalo.

Examples of other channel proteins include, but are not limited to:

ENaC/DEG family proteins including, but not limited to, SCNN1A, SCNN1B, SCNN1G, SCNN1D, ACCN2, ACCN1, ACCN3, ACCN4, and ACCN 5;

aquaporins (aquaponins) including, but not limited to, AQP1, AQP2, AQP3, AQP4, AQP5, AQP6, AQP7, AQP7P1, AQP7P2, AQP7P3, AQP7P4, AQP8, AQP9, AQP10, AQP11, AQP12A, and AQP 12B; and

chloride channels, including but not limited to CLCA1, CLCA2, CLCA3P, CLCA4, CLCC1, CLCF1, CLCN1, CLCN2, CLCN3, CLCN4, CLCN5, CLCN6, CLCN7, CLCNKA, and CLCNKB.

Examples of membrane carriers/transporters include, but are not limited to:

the ABCC family of proteins, including but not limited to ABCA, ABCA11, ABCA17, ABCB10P, ABCB, ABCC6P, ABCC, ABCD1P, ABCD, ABCE, ABCF, ABCG, TAP, CFTR TAPBP and TAPBPL;

The soluble carrier family of proteins includes, but is not limited to, SLC1A, SLC 2P, SLC2A3P, SLC2A4, SLC2A, SLC6A, SLC5A, SLC4A, SLC5A, SLC6A, SLC7A, SLC6A, SLC7A, SLC6A, SLC7A, SLC6A, SLC7A, SLC6, SLC12A, SLC13A, SLC14A, SLC15A, SLC16A, SLC25A, SLC22A, SLC25A, SLC22A, SLC25A, SLC22A, SLC25A, SLC22A, SLC25A, SLC22A, SLC25A, SLC22A, SLC25A, SLC22A, SLC25A, SLC22A, SLC25A, SLC22A, SLC25A, SLC26A, SLC27A, SLC28A, SLC29A, SLC30A, SLC35A, SLC38A, SLC35F, SLC35A, SLC38A, SLC35A, SLC38A, SLC35F, SLC35A, SLC38A, SLC35F, SLC38A, SLC35A, SLC38A, SLC35F, SLC38A, SLC35A, SLC38A, SLC35A, SLC38A, SLCO1B1, SLCO1B3, SLCO1C1, SLCO2A1, SLCO2B1, SLCO3A1, SLCO4A1, SLCO4C1, SLCO5A1, and SLCO6A 1;

ATP transporter proteins including, but not limited to, ATP1a1, ATP1a2, ATP1A3, ATP1a4, ATP1B3P 4, ATP1B4, ATP1L 4, ATP2a 4, ATP2B4, ATP2C 4, ATP4 4, ATP5a 4, ATP5AL 4, ATP5AP4, ATP5BL 4, ATP5C 4, ATP 5P 365 4, ATP 1P 366, ATP 365P 4, ATP 1P 366, ATP4, ATP 1P 366, ATP4, ATP 366P 366, ATP 366P 4, ATP 5P 366, ATP4, ATP 366, ATP 1P 366, ATP 366P 4, ATP 366P 366, ATP4, ATP 5P 366 4, ATP 366, ATP 366V 4, ATP 366 4, ATP 366P 366 4, ATP 366P 4, ATP 366 4, ATP 366 4, ATP 366P 4, ATP 366 4, ATP 366P 366 4, ATP 366 4, ATP 366 4, ATP 366 4, ATP 366 4, ATP 366 4, ATP 366P 366 4, ATP 366 4, ATP 366 4, ATP 366 36; and

fatty acid binding proteins, including but not limited to FABP1, FABP2, FABP3, FABP3P2, FABP4, FABP5, FABP5L1, FABP5L2, FABP5L3, FABP5L4, FABP5L5, FABP5L6, FABP5L7, FABP5L8, FABP5L9, FABP5L10, FABP5L11, FABP5L12, FABP6, FABP7, FABP9, and FABP 12;

Insulin-like growth factors, including but not limited to IGF1, IGF1R, IGF2, IGF2AS, IGF2BP1, IGF2BP2, IGF2BP3, IGF2R, IGFALS, IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6, IGFBP7, IGFBPL1, IGFL1, IGFL1P1, IGFL1P2, IGFL2, IGFL3, IGFL4, and IGFN 1;

transforming growth factors including, but not limited to, TGFA, TGFB1, TGFB1I1, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TGFBR3, TGFBRAP1, TGFBRE, LEFTY1, LEFTY2, BMPR1A, BMPR1APS1, BMPR1APS2, BMPR1B, BMPR2, ACVR1, ACVR1B, ACVR1C, ACVR2A, ACVR2B and ACVRL 1;

nuclear receptors including, but not limited to, NR1D1, NR1D2, NR1H2, NR1H3, NR1H4, NR1H5P, NR1I1, NR1I2, NR1I3, NR1I4, NR2a1, NR2a2, NR2B1, NR2B2, NR2B3, NR2C1, NR2C2 1, NR2E1, NR2F1, NR3C1, NR4a1, NR5a1, NR6a1, NRAP, NRBP, NRARP 0, NRIP1, NRBP1, NRIP1, NRBF 1, NR;

retinoic acid receptors including but not limited to RARA, RARB, RARG, RARRES1, RARRES2, and RARRES 3;

receptor tyrosine kinase orphan receptors and RAR-associated proteins, including but not limited to ROR1, ROR2, RORA, RORB, and RORC;

Peroxisome proliferator activated receptors including, but not limited to, PPARA, PPARD, PPARG, PPARGC1A, and PPARGC 1B;

thyroid hormone receptors including but not limited to THRA, THRAP3, THRAP3L, THRB and THRSP;

estrogen receptors, epithelial splice regulatory proteins, and estrogen related receptors including but not limited to ESR1, ESR2, ESRP1, ESRP2, ESRRA, ESRRAP1, ESRRAP2, ESRRB, and ESRRG;

erythroblastic leukemia virus oncogenes including, but not limited to, ERBB2, ERBB2IP, ERBB3, ERBB4, and EGFR;

platelet derived growth factors including but not limited to PDGFA, PDGFB, PDGFC, PDGFD, PDGFRA, PDGFRB and PDGFRL;

fibroblast-derived growth factors including, but not limited to, FGFR1, FGFR10P, FGFR10P2, FGFR2, FGFR3, FGFR3P, FGFR4, FGFR6, and FGFRL 1;

potential transforming growth factor beta binding proteins including, but not limited to, LTBP1, LTBP2, LTBP3, and LTBP 4;

vitamin carrier proteins, including but not limited to RBP1, RBP2, RBP3, RBP4, RBP5, RBP7, RBPJ, RBPJL, RBPJP1, RBPJP2, RBPJP3, RBPJP4, RBPMS2, and rbpmlps lp;

steroid hormone synthesis acute regulatory proteins including, but not limited to, STAR, STAR 3, STARD3NL, STARD4, STARD5, STARD6, STARD7, STARD8, STARD9, STARD10, STARD13, and STARP 1;

Sterol carrier proteins including, but not limited to, SCP2 and SCPEP 1;

glycolipid transporters including, but not limited to, GLTP, GLTPD1, GLTPD2, and GLTPP 1; and

other transporters such as CETP.

Other examples of proteins of interest include, but are not limited to:

t cell receptor beta constant 1, including but not limited to TRBC1, TRBC2, TRBD1, TRBD2, TRBJ1-1, TRBJ1-2, TRBJ1-3, TRBJ1-4, TRBJ1-5, TRBJ1-6, TRBJ2-1, TRBJ2-2 2-3, TRBJ2-4, TRBJ2-5, TRBJ2-6, TRBJ2-7, TRBV 2-1, TRBV 2-2, TR3672-3, TRBV 2-1, TRBV 2-2, TRBV 2-3, TRBV 2-4, TRBV-72-5, TRBV-72, TRBV 2-72, TRBV-72-2, TRBV-2-72, TRBV-72-2-72, TRBV-72, TRBV-72-2-3, TRBV-2-72-3, TRBV7-3, TRBV7-4, TRBV7-5, TRBV7-6, TRBV7-7, TRBV7-8, TRBV7-9, TRBV7-1, TRBV7-2, TRBV7-3, TRBV7-4, TRBV7-5, TRBV7-1, TRBV 20-7-2, TRBV7-1, TRBV 3621-7, TRBV 7-72-24, TRBV7-7, TRBV-7;

Resolvins including, but not limited to, ADAM3, ADAM5, ADAM21, ADAM DEC, ADAMTS TSL, ADAMTL, ADAMTS L, and ADATSL;

integrins including, but not limited to, ITGA1, ITGA2, ITGA2B, ITGA3, ITGA4, ITGA5, ITGA6, ITGA7, ITGA8, ITGA9, ITGA10, ITGA11, ITGAD, ITGAE, ITGAL, ITGAM, ITGAV, ITGAW, ITGAX, ITGB1, ITGB1BP1, ITGB1BP2, ITGB1BP3, ITGB2, ITGB3, ITGB3BP, ITGB4, ITGB5, ITGB6, ITGB7, ITGB8, and ITGBL 1;

cell attachment molecules including, but not limited to, NCAM1, NCAM2, VCAM1, ICAM1, ICAM2, ICAM3, ICAM4, ICAM5, PECAM1, L1CAM, CHL1, MAG, CADM1, CADM2, CADM3, and CADM 4;

human odorant receptors include, but are not limited to, OR10A, OR10C, OR10D, OR10G, OR10H, OR10J, OR10K, OR10Q, OR10R, OR10S, OR10T, OR10V, OR10Z, OR11A, OR11G, OR11H, OR11L, OR12D, OR13A, OR13C, OR13E, OR13F, OR13G, OR1J, OR 1H, OR2J, OR1F, OR2J, OR1D, OR2J, OR1F, OR1J, OR2D, OR1F, OR1J, OR2D, OR 1H, OR2J, OR 1H, OR14, OR1K, OR2K, OR10R, OR10H, OR10K, OR10H, OR11H, OR 1H, OR13C, OR 13R, OR13H, OR 1H, OR13H, OR 1H, OR2H, OR 1H, OR13C, OR13H, OR 13R 13C, OR13E 13C, OR 13R 13C, OR13E 13H, OR 1H, OR2D, OR 1H, OR13H, OR 1H, OR2D, OR13H, OR 13R 13H, OR1C, OR 13R 13, OR 13R 13, OR2F, OR2G, OR2H, OR2J, OR2K, OR2L, OR2M, OR2S, OR2T, OR2V, OR2W, OR2Y, OR2Z, OR3A, OR4B, OR4C, OR4D, OR4E, OR4D, OR4F, OR4K, OR4A, OR4K, OR52, OR4K, OR52, OR2T, OR2V, OR2W, OR2Y, OR2Z, OR3A, OR4K 51, OR52K, OR52, OR4A, OR52K 51, OR4A, OR4K 51, OR52K, OR4A, OR4K 51, OR4A, OR4K, OR4A, OR52, OR4K 51, OR4A, OR4K, OR4A, OR52, OR4K, OR52, OR4A, OR52, OR4K, OR4A, OR52, OR4A, OR4K, OR4A, OR4K 51, OR52, OR4K, OR4A, OR, OR56A, OR56B, OR5A, OR5AC, OR5AK, OR5AN, OR5AP, OR5AR, OR5AS, OR5AU, OR5B, OR5C, OR5D, OR5F, OR5G, OR5H, OR5I, OR5K, OR5L, OR5M, OR5P, OR5T, OR5V, OR6A, OR6B, OR6C, OR6F, OR6K, OR6P, OR5T, OR6V, OR6A, OR6B, OR6C, OR6F, OR6K 7K, OR 7H, OR7G 8G 7H, OR8G, OR 7H, OR7G 4, OR5H, OR7G, OR5H, OR7G 4, OR5L, OR 6G 4, OR7G, OR 6G 4, OR7G, OR 6G, OR7G 4, OR 6D, OR 6G, OR 7H, OR7G 4, OR7G, OR6C, OR7G, OR; and

Anopheles gambiae (anopheles gambiae) scent receptor including, but not limited to, IOR100, IOR101, IOR102, IOR103, IOR104, IOR105, IOR106, IOR107, IOR108, IOR109, IOR110, IOR111, IOR112, IOR113, IOR114, IOR115, IOR116, IOR117, IOR118, IOR119, IOR120, IOR121, IOR122, IOR123, IOR124, IOR125, IOR126, IOR127, IOR 7180, IOR 7097, IOR 7081, IOR 7080, IOR 7180, IOR 7097, ORL7080, ORL 7180, ORL7097, ORL7080, ORL7097, ORL7080, ORL 7180, ORL7097, ORL7080, ORL7097, ORL7080, ORL 7180, ORL7097, ORL7080, ORL 7180, ORL7080, ORL7097, ORL7080, ORL70, TPR 7116, ORL7117, ORL7118, ORL7119, ORL7120, ORL7121, ORL7122, ORL7123, ORL7124, ORL7125, TPR2307, TPR2308, TPR2309, TPR2310, TPR2312, TPR2314, TPR2315, TPR2316, TPR2317, TPR2318, TPR2319, TPR2320, TPR2321, TPR698, TPR699, TPR700, TPR701, TPR702, TPR716, TPR704, TPR706, TPR707, TPR719, TPR710, TPR711, TPR712, TPR713, TPR714, TPR715, TPR722, TPR717, TPR718, TPR720, TPR719, TPR762, TPR769, TPR739, TPR 73768, TPR 732049, TPR739, TPR769, TPR739, TPR 732049, TPR739, TPR 73.

Other examples of proteins of interest can be found in tables 7-22 below. In particular embodiments, spliced forms and/or SNPs of the proteins listed in tables 7-22 may be expressed. In particular embodiments, any combination of any of the proteins listed in tables 7-22 can be co-expressed in a cell.

In one aspect, the cells and cell lines of the invention are suitable for use in cell-based assays. Such cells and cell lines express proteins of interest consistently and reproducibly over time, and are therefore particularly advantageous in such assays.

In another aspect, the invention provides cells and cell lines suitable for the production of biomolecules. The cells and cell lines used for this purpose are characterized, for example, by the consistent expression of proteins or polypeptides that are functional or capable of becoming functional. The invention also provides cells and cell lines for producing stably expressed RNA or proteins of interest. By using the methods of the invention, cells and cell lines can be produced that express any desired protein in a functional form, including complex proteins such as multimeric proteins (e.g., heteromultimeric proteins) and cytotoxic proteins. The methods disclosed herein make it possible to generate engineered cells and cell lines that stably express functional proteins that were not generated prior to the present invention. Without being bound by theory, it is believed that because the method allows for the study of a very large number of cells or cell lines under any desired set of conditions, it makes it possible to identify rare cells that would otherwise not be able to be produced in a smaller population or otherwise discovered and are best suited to express the functional form of the desired protein under the desired conditions. Without being bound by theory, many RNAs and proteins of interest are normally expressed in specialized cells (e.g., cells of a particular tissue, cells of a specialized function, cells with specialized cellular regions or compartments, sensory cells, neurons, taste buds, epithelial cells, stem cells, cancer cells, muscle cells, cells of the eye, cells that produce antibodies, cells that produce high levels of protein, and the various cell types disclosed herein). Specialized cells may provide a specialized biological or cellular environment, background, or genetic makeup for non-cytotoxic or native functional or physiological expression of an RNA or protein of interest. For example, a specialized cell may provide factors, including co-factors or chaperones or specialized cellular compartments, for sufficient, proper or optimal expression, stoichiometry, yield, folding, assembly, post-translational modification, targeting, membrane integration, secretion, function, pharmacology, or physiology of an RNA or protein of interest. Engineering cells or cell lines to express their abnormally expressed RNA or protein of interest can result in the production of cells or cell lines under these conditions where optimal expression or function for the RNA or protein of interest is neither reproduced nor approached without associated cytotoxicity.

Many populations of cells that can be engineered to express an RNA or protein of interest consist of genetically diverse populations of individual cells in which even the number of chromosomes can vary from cell to cell. The rare cells comprised in these populations (as compared to the common cells of such populations) may provide a biological or cellular environment or background or genetic makeup that is sufficient, preferred, above average, improved or optimal for natural or non-cytotoxic expression, function, pharmacology or physiology of the RNA or protein of interest that is not normally expressed in the common cells of the cell population.

In certain embodiments, the present invention allows for the analysis of millions of individual cells of a population of cells engineered to contain an RNA or protein of interest, such that individual cells compatible with the expression of the RNA or protein of interest can be detected or isolated quickly or individually, even though this represents only rare cells in the population of cells. In certain embodiments, rare cells compatible with viable, non-cytotoxic, functional, or native expression of an RNA or protein of interest that typically causes cytotoxicity or cell death in a common cell of a population of cells engineered to express the RNA or protein of interest can be detected and isolated. In certain embodiments, according to the present invention, each positive cell detected or isolated from a population of cells engineered to express an RNA or protein of interest can comprise a different absolute or relative level of each RNA or protein of interest. In certain embodiments, each positive cell may also provide or comprise a different cellular or genetic background (e.g., a different number of chromosomes or chromosome segments, genes, gene sequences, expression profiles, or endogenously expressed proteins or RNAs (including mrnas or sirnas), or cofactors for an RNA or protein of interest) as a cellular background for the expression or function of the RNA or protein of interest. In certain embodiments, the invention provides for the isolation of a plurality of engineered cells positive for expression of an RNA or protein of interest coupled with a novel method capable of maintaining the isolated cells in culture. In certain embodiments, the maintenance of the isolated cells in culture may be performed using conditions that are substantially the same for all maintained cells. In certain embodiments, this in turn enables testing (including functional testing) of the isolated cells over time in culture to identify and produce functionally stable cells or cell lines comprising the desired or enhanced expression, function, physiology, or pharmacology of the expressed RNA or protein of interest. In certain embodiments, the methods allow for the identification or generation of cells that functionally, survivingly and stably express an RNA or protein of interest (even an RNA or protein of interest that is not normally expressed in normal cells of a population of cells engineered to express the RNA or protein of interest) by isolating, maintaining and functionally testing a number of cells that are positive for the expression of the RNA or protein of interest.

Indeed, in certain embodiments, the methods were found to result in the functional and stable expression of RNAs or proteins previously thought to be cytotoxic when expressed in engineered cells, demonstrating that the methods can be used to generate the efficacy of cells comprising conditions necessary and compatible with the non-cytotoxic expression and function of such proteins that previously could not be modeled in engineered cells.

In a further aspect, the invention provides a matched panel of cell lines, i.e. a collection of clonal cell lines matched for one or more physiological properties. Because the methods of the invention allow a large number of cell lines to be maintained and characterized under the same conditions, any number of cell lines with similar physiological properties can be identified. By using the method of the invention it is possible to prepare a matched set of subjects comprising any desired number of cell lines or to complement them. Such matched sets of subjects can be maintained under the same conditions (including cell density) and are therefore useful for high throughput screening and other uses where differences between cell lines need to be compared and identified. Also within the present invention is a matched panel of subjects for growth rate matched cell lines.

In another aspect, the invention provides methods for producing cells or cell lines that express proteins with previously unknown function and/or for which ligands have not been previously identified. Such proteins may be known naturally occurring proteins, previously unknown forms of known naturally occurring proteins, or modified forms of any of the foregoing.

Any desired cell type can be used in the cells of the invention. The cell may be a prokaryotic cell or a eukaryotic cell. The cells may or may not express the protein of interest in their native state. Eukaryotic cells that can be used include, but are not limited to, fungal cells such as yeast cells, plant cells, insect cells, and animal cells. Animal cells that can be used include, but are not limited to, mammalian cells. Primary or immortalized cells can be derived from the mesoderm, ectoderm or endoderm of eukaryotic organisms. The cell may be an endothelial cell, an epidermal cell, a mesenchymal cell, a neural cell, a renal cell, a hepatic cell, a hematopoietic cell, or an immune cell. For example, the cell may be an intestinal crypt or villus cell, a clara cell, a colon cell, an intestinal cell, a goblet cell, an enterochromaffin cell, an enteroendocrine cell. Mammalian cells useful in the present invention include, but are not limited to, humans, non-human primates, cows, horses, goats, sheep, pigs, rodents (including rats, mice, hamsters, guinea pigs), marsupials, rabbits, dogs, and cats. The cells may be differentiated cells or stem cells, including embryonic stem cells.

The cells of the invention may be cells of primary, transformed, oncogenically transformed, virally transformed, immortalized, conditionally transformed, explants, tissue-sliced cells, animals, plants, fungi, protists, archaebacteria and eubacteria, mammals, birds, fish, reptiles, amphibians and arthropods, birds, chickens, reptiles, amphibians, frogs, lizards, snakes, fish, worms, squid, lobsters, sea urchins, sea cucumbers, ascidians, flies, squid, arthropods, beetles, chickens, lampreys, rice (ricefish), zebra finches, blowfish and zebrafish.

Furthermore, cells such as blood/immune cells, endocrine (thyroid, parathyroid, adrenal), GI (mouth, stomach, intestine), liver, pancreas, gall bladder, respiratory tract (lung, trachea, pharynx), cartilage, bone, muscle, skin, hair, urinary tract (kidney, bladder), reproduction (sperm, ovum, testis, uterus, ovary, penis, vagina), sense (eye, ear, nose, mouth, tongue, sensory neurons), blood/immune cells such as B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, T helper cells, γ δ T cells, natural killer cells, granulocytes (basophils, eosinophils, neutrophils/multilobal neutrophils), monocytes/macrophages, red blood cells (reticulocytes), mast cells, liver, pancreas, gall bladder, liver, and/or kidney, Thrombocytes/megakaryocytes, dendritic cells; endocrine cells such as thyroid (thyroid epithelial cells, parafollicular cells), parathyroid (parathyroid chief cells, eosinophils), adrenal (pheochromocytes), nervous system cells such as: glial cells (astrocytes, microglia), large cell neurosecretory cells, astrocytes, nuclear chain cells, burtther cells, pituitary cells (gonadotropic cells, corticotropin cells, thyroid stimulating cells, parental cells, prolactin cells), respiratory system cells such as lung cells (type I lung cells, type II lung cells), clara cells, goblet cells; circulating system cells such as cardiomyocytes, pericytes; digestive system cells such as stomach (stomach chief cell, stomach parietal cell), goblet cell, panne cell, G cell, D cell, ECL cell, I cell, K cell, enteroendocrine cell, enterochromaffin cell, APUD cell, liver (hepatocyte, kupffer cell), pancreas (β cell, α cell), gall bladder; cells of the cartilage/bone/muscle/integumentary system such as osteoblasts, osteocytes, osteoclasts, dental cells (cementoblasts, ameloblasts), chondrocytes: chondroblasts, chondrocytes, skin/hair cells: silk cells (trichocytes), keratinocytes, melanocytes, muscle cells: myocytes, adipocytes, fibroblasts, urinary system cells such as podocytes, juxtaglomerular cells, mesangial cells/extrabulbar cells, proximal tubular brush border cells, dense plaque cells; reproductive system cells such as sperm, sertoli cells, leydig cells, ova, follicular cells; sensory cells such as cells of the organ of corti, olfactory epithelium, temperature sensitive sensory neurons, merkel cells, olfactory receptor neurons, pain sensitive neurons, photoreceptor cells, taste bud cells, hair cells of the vestibular apparatus, carotid body cells are used to prepare the cells or cell lines of the invention.

Useful plant cells include root, stem and leaf cells, plant tissues include meristems, parenchyma, horny tissue, sclerenchyma, secretory tissue, xylem, phloem, epidermis, periderm (bark).

Cells for use in the cells and cell lines of the invention also include, but are not limited to: chinese Hamster Ovary (CHO) cells, defined neuronal cell lines, pheochromocytoma, neuroblastoma, fibroblast, rhabdomyosarcoma, dorsal root ganglion cells, NS0 cells, CV-1(ATCC CCL70), COS-1(ATCC CRL 1650), COS-7(ATCC CRL 1651), CHO-K1(ATCC CCL 61), 3T3(ATCC CCL 92), NIH/3T3(ATCC CRL1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCCCL 26), MRC-5(ATCC CCL 171), L-cells, HEK-293(ATCC CRL1573) and PC12(ATCC CRL 1721), HEK293T (ATCC CRL-11268), RBATCL (ATCC CCRL-1378), SH-SY 5(ATCC CRL-5Y), ATCC 226K (ATCC CRL-22634), ATCC-30 (SJ-861) CRL 30(ATCC CRL-11268), RBATCCRL-2065 (ATCC CCL-Y), HepG2(ATCC HB-8065), ND7/23(ECACC92090903), CHO (ECACC 85050302), Vero (ATCC CCL 81), Caco-2(ATCC HTB 37), K562(ATCC CCL 243), Jurkat (ATCCIB-152), Per.C6(Crucell, Leiden, The Netherlands), huvec (ATCC human primary PCS 100-010, mouse CRL 2514, CRL 2515, CRL 2516), HuH-7D12(ECACC 01042712), 293(ATCC CRL 10852), A549(ATCC CCCL 185), IMR-90(ATCC CCL 186), MCF-7(ATC HTB-22), U-2OS (ATCC HTB-96), T84(ATCC CCL 248) or any defined cell line (polarized or non-polarized) or any cell line obtainable from a repository such as the American type culture center (ATCC, 10801University Blvd. Manassas, Va.20110-2209USA) or the European cell culture center (ECACC, Salisbury Wiltshire SP40 JGEngland).

Furthermore, the cells used in the method of the invention are mammalian cells that are susceptible to growth in a serum-containing medium, a serum-free medium, a fully known medium without any animal-derived products, and cells that can be switched from one of such conditions to another.

The cells of the invention include cells into which a nucleic acid encoding a protein of interest (or, in the case of a heteromultimeric protein, a nucleic acid encoding one or more subunits of the protein) has been introduced. Engineered cells also include cells into which has been introduced a nucleic acid for transcriptional activation of an endogenous sequence encoding a protein of interest (or for transcriptional activation of an endogenous sequence encoding one or more subunits of a heteromultimeric protein). Engineered cells also include cells comprising a nucleic acid encoding a protein of interest that can be activated by contact with an activating compound or after post-translational modification, or after treatment with or contact with an enzyme (including but not limited to a protease). Engineered cells also include combinations of the above, i.e., cells that express one or more subunits of heteromultimeric proteins from introduced nucleic acids encoding them and that express one or more subunits of proteins by gene activation.

Any nucleic acid can be introduced into a cell using known methods. Techniques for introducing nucleic acids into cells are well known and can be readily understood by one of ordinary skill in the art. Such methods include, but are not limited to, transfection, viral delivery, protein or peptide mediated insertion, co-precipitation methods, lipid-based delivery reagents (lipofection), cytofectins, lipopolyamine delivery, dendrimer delivery, electroporation, or mechanical delivery. Examples of transfection agents are GENEPORTER, GENEPORTER2, LIPOFECTANCE 2000, FUGENENE 6, FUGENENE HD, TFX-10, TFX-20, TFX-50, OLIGOFECTAMINE, TRANSFAST, TRANSFECTAM, GENESHUTTLE, TROXENE, GENESILENCER, X-TREMEGENE, PERFECTIN, CYTOFECTIN, SIPORT, UNIFECTOR, SIFECTOR, TRANSIT-LT1, TRANSIT-LT2, TRANSIT-EXPRESS, IFECT, RNASITTLE, METAFACTENE, LYOVEC, LIPOTAXI, GENEERASER, GENEJUICE, CYTOPURE, JEI, JETPTPT, MEGACTIN, POLYFECTT, TRANSSANGER, RNAFEFEFEFEFEFEFECTT, FECTEFE, PEI-FECTF, CTIN, and CLOFECTE

When two or more nucleotide sequences are introduced, e.g., sequences encoding two or more subunits of a heteromultimeric protein or sequences encoding two or more different proteins of interest, the sequences can be introduced on the same vector, or preferably on separate vectors. The DNA may be genomic DNA, cDNA, synthetic DNA, or a mixture thereof. In certain embodiments, the nucleic acid encoding the protein of interest or a portion of the protein of interest does not include additional sequences, such that a protein is expressed that has additional amino acids that alter the function of the cell compared to the physiological function of the protein.

In certain embodiments, the nucleic acid encoding the protein of interest comprises one or more substitutions, insertions, mutations, or deletions compared to the nucleic acid sequence encoding the wild-type protein. In embodiments that include nucleic acids comprising mutations, the mutations can be random mutations or site-directed mutations. Such nucleic acid changes may or may not result in amino acid substitutions. In certain embodiments, the nucleic acid is a fragment of a nucleic acid encoding a protein of interest. Nucleic acids that are fragments or have such modifications encode polypeptides that retain at least one biological property of the protein of interest.

The invention also includes cells and cell lines that stably express: a nucleic acid whose sequence is at least about 85% identical to a "wild-type" sequence encoding a protein of interest, or a corresponding nucleic acid derived from a species other than human, or a nucleic acid encoding an amino acid sequence identical to any of those nucleic acids. In certain embodiments, the sequence identity is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher compared to those sequences. The invention also includes cells and cell lines in which a nucleic acid encoding a protein of interest hybridizes under stringent conditions to the wild-type sequence, or to a corresponding nucleic acid derived from a species other than human, or to a nucleic acid encoding the same amino acid sequence as any of those nucleic acids.

In certain embodiments, the cell or cell line comprises a nucleic acid sequence encoding a protein, which nucleic acid sequence comprises at least one substitution, as compared to the wild-type sequence, or a corresponding nucleic acid derived from a species other than human, or a nucleic acid encoding the same amino acid sequence as any of those nucleic acids. Substitutions may include less than 10, 20, 30 or 40 nucleotides, or up to or equal to 1%, 5%, 10% or 20% of the nucleotide sequence. In certain embodiments, the sequence that is replaced may be substantially identical to (e.g., a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to) the wild-type sequence, or a corresponding nucleic acid derived from a species other than human, or a nucleic acid encoding an amino acid sequence identical to any of those nucleic acids, or a sequence that is capable of hybridizing under stringent conditions to the wild-type sequence, or a corresponding nucleic acid derived from a species other than human, or a nucleic acid encoding an amino acid sequence identical to any of those nucleic acids.

In certain embodiments, the cell or cell line comprises a nucleic acid sequence encoding a protein, which nucleic acid sequence comprises an insertion or deletion of a nucleic acid from a wild-type sequence, or a corresponding nucleic acid derived from a species other than human, or a nucleic acid encoding the same amino acid sequence as any of those nucleic acids. Insertions or deletions may be of less than 10, 20, 30 or 40 nucleotides, or up to or equal to 1%, 5%, 10% or 20% of the nucleotide sequence. In certain embodiments, the inserted or deleted sequence may be substantially identical to (e.g., a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to) the wild-type sequence, or a corresponding nucleic acid derived from a species other than human, or a nucleic acid encoding an amino acid sequence identical to any of those nucleic acids, or a sequence capable of hybridizing under stringent conditions to the wild-type sequence, or a corresponding nucleic acid derived from a species other than human, or a nucleic acid encoding an amino acid sequence identical to any of those nucleic acids.

In certain embodiments, the nucleic acid substitution or modification results in an amino acid change, such as an amino acid substitution. For example, an amino acid residue of a wild-type protein of interest or a corresponding amino acid derived from a species other than human may be replaced by conservative or non-conservative substitutions. In certain embodiments, the sequence identity between the original amino acid sequence and the modified amino acid sequence may differ by about 1%, 5%, 10%, or 20%, or differ from a sequence substantially identical thereto (e.g., a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto).

A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity) as the parent amino acid residue. In cases where 2 or more amino acid sequences differ from each other by conservative substitutions, the percentage of sequence identity or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Methods for making such adjustments are well known to those skilled in the art. See, e.g., Pearson, Methods mol. biol. 243: 307-31(1994).

Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chain: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy side chain: serine and threonine; 3) amide-containing side chain: asparagine and glutamine; 4) aromatic side chain: phenylalanine, tyrosine and tryptophan; 5) basic side chain: lysine, arginine and histidine; 6) acidic side chain: aspartic acid and glutamic acid; and 7) sulfur containing side chains: cysteine and methionine. Preferred conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate and asparagine-glutamine. Alternatively, conservative amino acid substitutions are described in Gonnet et al, Science 256: any change in the PAM250 log likelihood matrix with positive values as disclosed in 1443-45 (1992). A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log likelihood matrix.

Conservative modifications in a protein of interest will result in a protein having functional and chemical characteristics that are similar (i.e., at least 50%, 60%, 70%, 80%, 90%, or 95% identical) to the functional and chemical characteristics of the unmodified protein.

In one embodiment, the host cell is an embryonic stem cell, which is then used as the basis for the production of transgenic animals producing the protein of interest. Embryonic stem cells that stably express a functional protein of interest can be implanted directly into an organism, or their nuclei can be transferred into other recipient cells, which can then be implanted, or they can be used to produce transgenic animals. In certain embodiments, the protein may be expressed in an animal with desired transient and/or tissue-specific expression.

As will be appreciated by those of ordinary skill in the art, any vector suitable for use in a selected host cell may be used to introduce a nucleic acid encoding a protein of interest into the host cell. When more than one vector is used, for example, to introduce two or more different subunits or two or more proteins of interest, the vectors may be of the same type or may be of different types.

Examples of vectors that can be used to introduce the nucleic acid into the host cell include, but are not limited to, plasmids, viruses (including retroviruses and lentiviruses), cosmids, artificial chromosomes, which can include, for example, pCMVScript, pcDNA3.1Hygro, pcDNA3.1neo, pcDNA3.1puro, pSV2neo, pIRES puro, pS V2 zeo. Exemplary mammalian expression vectors that can be used to generate the cells and cell lines of the invention include: pFN11A (BIND)pGL4.31、pFC14A(7)CMVpFC14K(7)CMVpFN24A(7)CMVd3pFN24K(7)CMVd3HaloTag^TM pHT2、pACT、pAdVAntage^TM、pBIND、An enhancer,Promoter, pCI, pCMVTNT^TM、pG5luc、pSI、pTARGET^TM、pTNT^TM、pF12A RMpF12K RMpRegneo、pYES2/GS、pAd/CMV/V5-DESTVector, pAd/PL-DEST^TMA carrier,pDEST^TM27 carrier, a,pEF-DEST51 vector,pcDNA^TMDEST47 vector, pCMV/Bsd vector, pEF6/HisA, B and C, pcDNA^TM2-DEST, pLenti6/TR, pLP-AcGFP1-C, pLPS-AcGFP1-N, pLP-IRESNeo, pLP-TRE2, pLP-RevTRE, pLP-LNCX, pLP-CMV-HA, pLP-CMV-Myc, pLP-RetroQ and pLP-CMVneo.

In certain embodiments, the vector comprises an expression control sequence such as a constitutive or conditional promoter, preferably a constitutive promoter is used. One of ordinary skill in the art will be able to select such sequences. For example, suitable promoters include, but are not limited to, CMV, TK, SV40, and EF-1 α. In certain embodiments, the promoter is an inducible promoter, a temperature regulated promoter, a tissue specific promoter, a repressible promoter, a heat shock type promoter, a developmental type promoter, a cell lineage specific promoter, a eukaryotic promoter, a prokaryotic promoter, or a transient promoter, or a combination or recombination of unmodified or mutagenized, randomized, shuffled sequences of any one or more of the above. In other embodiments, the protein of interest is expressed by gene activation or episome.

In certain embodiments, the vector lacks a selectable marker or drug resistance gene. In other embodiments, the vector optionally comprises a nucleic acid encoding a selectable marker (e.g., a protein that confers drug or antibiotic resistance or, more commonly, any product that exerts selective pressure on the cell). When more than one vector is used, each vector may have the same or different drug resistance or other selection pressure markers. If more than one drug resistance or the selection pressure markers are the same, simultaneous selection can be achieved by increasing the level of the drug. Suitable markers are well known to those of ordinary skill in the art and include, but are not limited to, polypeptide products that confer resistance to any of the following: neomycin/G418, puromycin, hygromycin, bleomycin (Zeocin), methotrexate and blasticidin. Although drug selection (or selection using any other suitable selection marker) is not a necessary step in the generation of the cells and cell lines of the invention, it can be used to enrich the transfected cell population for stably transfected cells, provided that the transfected construct is designed to confer drug resistance. If a signaling probe is used for subsequent selection of cells expressing the protein of interest, premature selection after transfection may result in some positive cells that may be only transiently transfected and not stably transfected. However, this effect can be minimized by passaging sufficient cells to allow transient expression in the diluted transfected cells.

A variety of compounds or treatments known to one of ordinary skill in the art can be used to apply selective pressure to the cells. Without being bound by theory, selective pressure may be applied by contacting the cells with conditions that are suboptimal or detrimental to cell growth, progression or viability of the cell cycle, such that cells that are resistant or resistant to these conditions are selected as compared to cells that are not resistant or resistant to these conditions. Conditions that may be used to apply or administer selective pressure include, but are not limited to, antibiotics that slow or prevent cell growth or synthesis of biological building blocks, drugs, mutagens, compounds that interrupt RNA, DNA, or protein synthesis, removal or limitation of nutrients, amino acids, carbohydrates, or compounds necessary for cell growth or viability, treatment, e.g., growth or maintenance of cells under conditions that are suboptimal for cell growth, e.g., at suboptimal temperatures, atmospheric conditions (e.g., percentage of carbon dioxide, oxygen, or nitrogen or humidity), or under nutrient-poor media conditions. Without being bound by theory, selection pressure may be used to select for markers, factors or genes that confer or encode resistance or tolerance to the selection pressure. For example, (i) a population of cells may first be contacted with or introduced into the cells with such a marker, factor or gene that confers resistance or tolerance to a selective pressure such that each cell can assimilate or be modified to comprise a different level or not of the marker, factor or gene, and (ii) the population may then be contacted with a selective pressure to which the marker, factor or gene confers resistance or tolerance such that the cells comprising the marker, factor or gene comprise a growth advantage over the cells that do not comprise the marker, factor or gene. Without being bound by theory, cells comprising elevated levels of markers, factors or genes will show proportionally increased tolerance to the corresponding selection pressure. Selection pressure can be used to select cells comprising a desired property, an RNA or protein of interest associated with said property, an RNA or protein having a marker, factor or gene conferring tolerance or resistance to the respective selection pressure. Without being bound by theory, cells having a proportionally elevated level of a desired property, RNA or protein of interest can be selected by administering a proportionally increased level or amount of selection pressure during the selection process. If cells comprising multiple properties, RNAs or proteins are desired, each of such properties, RNAs or proteins may be associated with a relevant marker, factor or gene conferring resistance to the same or different forms of selection pressure, and selection using all such selection pressures may be used to select cells comprising all of the desired properties, RNAs or proteins of interest. After selecting cells having a desired property, RNA or protein of interest, the selected cells can be maintained at the same, increased or decreased level, concentration, dose, or treatment of the selection pressure used during the selection process. In some cases, periodic increases in the level, concentration, dose, or treatment of the selection pressure can be used to select cells that expand the desired property, RNA, or protein of interest, at correspondingly increasingly elevated levels. In some cases, after selecting cells using a selection pressure, the selected cells are maintained using a reduced level, concentration, dose, or treatment of the selection pressure to help ensure that the selected desired properties, RNA, or protein are maintained in the maintained cells.

The level of selection pressure used can be determined by one of ordinary skill in the art. This can be done, for example, by performing a sterilization curve experiment (kill curve experiment) in which control cells and cells comprising a resistance marker, factor or gene are tested at increasing levels, doses, concentrations or selection pressure of treatment and time ranges for negative cell selection, only within or preferentially over the desired time range (e.g., 1 to 24 hours, 1 to 3 days, 3 to 5 days, 4 to 7 days, 5 to 14 days, 1 to 3 weeks, 2 to 6 weeks, 1 to 2 months, 1 to 3 months, 4, 5, 6, 7, 8, 9 or more than 10 months). The exact level, concentration, dose or treatment of the selection pressure that can be used depends on the cell used, the desired property itself, the marker, factor or gene conferring resistance or tolerance to the selection pressure, and the level of the desired property desired in the selected cell, and one of ordinary skill in the art can readily understand how to determine the appropriate range based on these considerations. In some cases, after selection, a selection pressure of less than 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the levels, concentrations, doses, or treatments used in the selection process is used for subsequent maintenance of the selected cells. In some cases where multiple different selection pressures are used, the level, concentration, dose, or treatment of each selection pressure used in the selection process may be reduced in subsequent maintenance of the selected cells, e.g., to less than 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of that used in the selection process itself. In certain embodiments, such reduced levels, concentrations, doses, or treatments are selected such that cells selected to comprise the desired property, RNA, or protein continue to comprise the desired property over time in culture. In certain embodiments, at most the level, concentration, dose, or treatment necessary to prevent loss or reduction of the desired property in the selected cells is used during the period of maintaining the cells in culture, e.g., to minimize exposure of the cells to any possible effect detrimental to the cells resulting from the use of a higher level, concentration, dose, or treatment than is necessary for the cells to retain the property, RNA, or protein according to which the cells are selected.

In certain embodiments, the cells and cell lines of the invention are capable of retaining the property, RNA or protein against which the cells are selected (e.g., expression of the protein or RNA of interest) in the absence of selective pressure for at least 15 days, 30 days, 45 days, 60 days, 75 days, 100 days, 120 or 150 days. Such cells and cell lines may be cultured in the presence of the same, increased or decreased levels, concentrations, doses or treatment of the selection pressure as compared to the levels, concentrations, doses or treatment of the selection pressure used in the selection process. Such cells and cell lines can be cultured in the absence of any selective pressure. In the case where the level, concentration, dose or treatment is reduced, they may be reduced to less than 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the level, concentration, dose or treatment, respectively, used in the selection process. In cases where cells and cell lines express more than one protein or RNA of interest, and the expression of each protein or RNA of interest is selected using different selection pressures, the level, concentration, dose, or treatment of each selection pressure can be independently selected during the culture of the cells and cell lines after selection, e.g., each selection pressure can be independently selected to be absent in the cell culture, or at the same or an increased or decreased level, concentration, dose, or treatment as compared to its respective level, concentration, dose, or treatment used during selection. In the case where the levels, concentrations, doses or treatments are reduced, they may be reduced to 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the respective levels, concentrations, doses or treatments used in the selection process.

In certain embodiments, the nucleic acid sequence encoding the protein further comprises a tag. Such tags may encode, for example, a HIS tag, a myc tag, a Hemagglutinin (HA) tag, proteins C, VSV-G, FLU, Yellow Fluorescent Protein (YFP), green fluorescent protein, FLAG, BCCP, a maltose binding protein tag, a Nus-tag, Softag-1, Softag-2, a Strep-tag, an S-tag, thioredoxin, GST, V5, TAP, or CBP. The tags can be used to determine protein expression levels, intracellular localization, protein-protein interactions, regulation of a protein of interest, or function of a protein. Tags may also be used to purify or fractionate proteins.

In the case of cells and cell lines expressing RNA of interest, the RNA can be any type of RNA, including antisense RNA, short interfering RNA (sirna), transfer RNA (trna), structural RNA, ribosomal RNA, heterogeneous nuclear RNA (hnrna), and small nuclear RNA (snrna), messenger RNA (mrna), RNA in a stem-loop structure, RNA in a hairpin structure, RNA comprising single-stranded RNA, RNA comprising double-stranded RNA, RNA that binds a protein, RNA that binds a fluorescent compound, RNA having biological activity, RNA encoding a product of biological activity, catalytic RNA, RNA oligonucleotides, RNA that can mediate RNAi, or RNA that can modulate the level or activity of at least a second RNA.

In embodiments in which the cells and cell lines of the invention express a functional protein of interest, the protein may be any protein, including but not limited to single chain proteins, multi-chain proteins, heteromultimeric proteins. In the case of multimeric proteins, in certain embodiments, the cell is expressing all of the subunits that make up the native protein. The protein may have a "wild-type" sequence or may be a variant. In certain embodiments, the cell expresses a protein comprising variants of one or more subunits, including allelic variants, splice variants, truncated variants, isoforms, subunits of different stoichiometries, different assemblies of subunits, forms that fold differentially, forms that are differentially active, forms with different functionalities, forms with different binding properties, forms that associate with different cofactors, forms that are expressed in different cellular contexts, forms that are expressed in different cellular genetic contexts, forms that are expressed in cells with different endogenous expression profiles, forms that are differentially localized, chimeric forms or chemically modified forms, enzymatically modified forms, post-translationally modified forms, glycosylated forms, proteolytic forms, chimeric subunits, and mutant forms comprising amino acid substitutions (conserved or non-conserved), modified amino acids (including chemically modified amino acids), and non-naturally occurring amino acids And combinations thereof. The heteromultimeric proteins expressed by the cells or cell lines of the invention may comprise subunits from 2 or more species, for example subunits from species homologues of the protein of interest.

In certain embodiments, the cells of the invention express two or more functional proteins of interest. According to the present invention, such expression may result from the introduction of a nucleic acid encoding all or part of the protein of interest, from the introduction of a nucleic acid that activates transcription of all or part of the protein of interest from an endogenous sequence, or from any combination thereof. The cells can express any desired amount of the protein of interest. In various embodiments, the cell expresses 3, 4, 5, 6 or more proteins of interest. For example, the invention includes cells and cell lines that stably express functional proteins in a pathway of interest or proteins from cross-pathways (including enzymatic pathways, signaling pathway regulatory pathways, etc.). In certain embodiments, the cells or cell lines of the invention stably express one or more functional RNAs and/or proteins involved in a biological pathway of interest, e.g., a protein and/or RNA component of a biological pathway and a protein and/or RNA modulator of a biological pathway and/or one or more components thereof. In certain embodiments, a biological pathway consists of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or at least 50 protein components. In certain embodiments, the biological pathway consists of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or at least 50 RNA components. Examples of biological pathways in which functional proteins may be stably expressed by the cells and cell lines of the invention include, but are not limited to: 2-arachidonoylglycerol biosynthetic pathway, 5-hydroxytryptamine degradation pathway, receptor type 5ht1 mediated signaling pathway, receptor type 5ht2 mediated signaling pathway, receptor type 5ht3 mediated signaling pathway, receptor type 5ht4 mediated signaling pathway, acetate utilization pathway, adenine and hypoxanthine salvage pathway, epinephrine and norepinephrine biosynthetic pathway, alanine biosynthetic pathway, allantoin degradation pathway, alpha adrenergic receptor signaling pathway, Alzheimer's disease-amyloid protein secretase pathway, Alzheimer's disease-presenilin pathway, aminobutyric acid degradation pathway, arachidonoylethanolamide biosynthetic pathway, arachidonoylethanolamide degradation pathway, androgen/estrogen/progesterone biosynthetic pathway, An angiogenic pathway, an angiotensin ii-stimulated signaling pathway by G-protein and beta-arrestin, an apoptotic signaling pathway, an arginine biosynthetic pathway, an ascorbate degradation pathway, an asparagine and aspartate biosynthetic pathway, an ATP synthetic pathway, axonal guidance mediated by nerve growth factor, axonal guidance mediated by signallings, axonal guidance mediated by slit/robo, a B cell activation pathway, a beta adrenergic receptor signaling pathway, a beta 2 adrenergic receptor signaling pathway, a beta 3 adrenergic receptor signaling pathway, a biotin biosynthetic pathway, a blood clotting pathway, an bupropion degradation pathway, a cadherin signaling pathway, a carnitine metabolic pathway, a cell cycle pathway, a cholesterol biosynthetic pathway, a chorismate biosynthetic pathway, a, Circadian clock system pathway, cobalamin biosynthesis pathway, coenzyme A binding linked carnitine metabolic pathway, corticotropin releasing factor receptor signaling pathway, cysteine biosynthesis pathway, cytoskeletal regulation by rho GTPase, neopurine biosynthesis pathway, neopyrimidine deoxyribonucleotide biosynthesis pathway, neopyrimidine ribonucleotide biosynthesis pathway, DNA replication, dopamine receptor mediated signaling pathway, EGF receptor signaling pathway, endogenous cannabinoid signaling pathway, endothelin signaling pathway, enkephalin release pathway, fas signaling pathway, fgf signaling pathway, flavin biosynthesis pathway, formyl tetrahydroformate biosynthesis pathway, fructose galactose metabolism pathway, GABA-b receptor ii signaling pathway, gamma-aminobutyric acid synthesis pathway, and the like, General transcription by RNA polymerase I, general transcriptional regulation, glutamine glutamate conversion pathway, glycolysis pathway, hedgehog signaling pathway, heme biosynthetic pathway, heterotrimeric G-protein signaling pathway-gi α and gs α mediated pathway, heterotrimeric G-protein signaling pathway-gq α and go α mediated pathway, heterotrimeric G-protein signaling pathway-extrarod light transduction pathway, histamine h1 receptor mediated signaling pathway, histamine h2 receptor mediated signaling pathway, histamine synthesis pathway, histidine biosynthetic pathway, Huntington's disease, hypoxia response by hif activation, inflammation mediated by chemokine and cytokine signaling pathways, insulin/igf pathway-mitogen activated protein kinase/map kinase cascade, Insulin/igf pathway-protein kinase B signaling cascade, integrin signaling pathway, interferon-gamma signaling pathway, interleukin signaling pathway, ionotropic glutamate receptor pathway, isoleucine biosynthetic pathway, jak/stat signaling pathway, leucine biosynthetic pathway, lipoic acid biosynthetic pathway, lysine biosynthetic pathway, mannose metabolic pathway, metabotropic glutamate receptor group i pathway, metabotropic glutamate receptor group ii pathway, metabotropic glutamate receptor group iii pathway, methionine biosynthetic pathway, methyl citrate cycle, methylmalonate pathway, mRNA splicing, muscarinic acetylcholine receptor 1 and 3 signaling pathways, muscarinic acetylcholine receptor 2 and 4 signaling pathways, n-acetylglucosamine metabolism, nicotine degradation pathway, and the like, Nicotinic acetylcholine receptor signaling pathway, notch signaling pathway, o-antigen biosynthetic pathway, proopiod dynorphin pathway, opioid proenkephalin pathway, opioid proopiomelanocortin pathway, ornithine degradation pathway, oxidative stress response pathway, oxytocin receptor-mediated signaling pathway, p38mapk pathway, p53 pathway, p53 pathway by glucose deprivation, p53 pathway feedback loop 1, p53 pathway feedback loop 2, pantothenic acid biosynthetic pathway, parkinson's disease, PDGF signaling pathway, pentose phosphate pathway, peptidoglycan biosynthetic pathway, phenyl acetate degradation pathway, phenylalanine biosynthetic pathway, benzene degradation pathway, phenylpropionate degradation pathway, pi3 kinase pathway, plasminogen activation cascade, proline biosynthetic pathway, prpp biosynthetic pathway, purine metabolism, Pyridoxal phosphate salvage pathway, pyridoxal 5-phosphate biosynthesis pathway, pyrimidine metabolism, pyruvate metabolism, ras pathway, s-adenosylmethionine biosynthesis pathway, salvage of pyrimidine deoxyribonucleotides, salvage of pyrimidine ribonucleotides, serine glycine biosynthesis pathway, succinate to propionate conversion, sulfate assimilation pathway, synaptic vesicle trafficking, T cell activation pathway, TCA cycle, tetrahydrofolate biosynthesis pathway, TGF- β signaling pathway, thiamine biosynthesis pathway, thiamine metabolism, threonine biosynthesis pathway, thyroid stimulating hormone releasing hormone receptor signaling pathway, toll receptor signaling pathway, transcriptional regulation by bzip transcription factor, triacylglycerol metabolism, tryptophan biosynthesis pathway, tyrosine biosynthesis pathway, ubiquitin proteasome pathway, enzyme activity, protein metabolism, protein, A valine biosynthetic pathway, a vasopressin synthetic pathway, a VEGF signaling pathway, a vitamin B6 biosynthetic pathway, a vitamin B6 metabolism, a vitamin D metabolism and pathway, a wnt signaling pathway, and a xanthine and guanine salvage pathway.

Other examples of biological pathways in which functional proteins can be stably expressed by the cells and cell lines of the invention include, but are not limited to, the following biological processes: amino acid metabolism (e.g., amino acid biosynthesis, amino acid catabolism, regulation of amino acid metabolism, amino acid transport, and other amino acid metabolism), transport (e.g., amino acid transport, carbohydrate transport, vitamin/cofactor transport, anion transport, cation transport, lipid and fatty acid transport, nucleoside, nucleotide and nucleic acid transport, phosphate transport, extracellular transport and import, small molecule transport, and other transport), apoptosis (e.g., induction of apoptosis, inhibition of apoptosis, other apoptosis, and other apoptotic processes), blood circulation and gas exchange, carbohydrate metabolism (e.g., carbohydrate transport, disaccharide metabolism, gluconeogenesis, glycogen metabolism, glycolysis, monosaccharide metabolism, other carbohydrate metabolism, other polysaccharide metabolism, pentose phosphate branch, carbohydrate metabolism, and regulation of tricarboxylic acid pathways) Cell adhesion, cell cycle (e.g., cell cycle control, DNA replication, mitosis, and other cell cycle processes), cell proliferation and differentiation, cell structure and motility, coenzyme and prosthetic metabolism (e.g., coenzyme metabolism, porphyrin metabolism, pterin metabolism, vitamin/cofactor transport, vitamin biosynthesis, vitamin catabolism, and other coenzyme and prosthetic metabolism), developmental processes (e.g., ectodermal development, anterior axis establishment/posterior axis establishment, determination of the dorsal/ventral axis, embryogenesis, endodermal development, fertilization, meiosis, mesodermal development, segment specification, sex determination, oogenesis, spermatogenesis and motility, and other developmental processes), electron transport (e.g., ferredoxin metabolism, oxidative phosphorylation, and other electron transport pathways), homeostasis (calcium homeostasis), and homeostasis (e.g., calcium homeostasis), Glucose homeostasis, growth factor homeostasis, and other homeostasis activities), immunity and defense (e.g., antioxidant action and free radical scavenging, B cell-and antibody-mediated immunity, blood clotting, complement-mediated immunity, cytokine/chemokine-mediated immunity, detoxification, granulocyte-mediated immunity, interferon-mediated immunity, macrophage-mediated immunity, natural killer cell-mediated immunity, stress responses, T cell-mediated immunity, and other immune and defense approaches), intracellular protein trafficking (e.g., exocytosis, endocytosis, general vesicle trafficking, lysosomal trafficking, mitochondrial trafficking, peroxisome trafficking, and other intracellular protein trafficking), lipid, fatty acid, and steroid metabolism (e.g., acyl-Coa metabolism, fatty acid beta-oxidation, beta-lipoxygenase activity, beta-lipoxygenase, and other immune and defense activities), immune responses (e.g., immune responses, and free radical scavenging, B cell-and, Fatty acid biosynthesis, fatty acid desaturation, lipid and fatty acid binding, lipid and fatty acid transport, lipid metabolism and other lipids, fatty acid and steroid metabolism, phospholipid metabolism, lipid regulation, fatty acid and steroid metabolism, bile acid metabolism, cholesterol metabolism, steroid hormone metabolism and other steroid metabolism), muscle contraction, neuronal activity (e.g., action potential transmission, neuro-neurosynaptic transmission, neuromuscular synaptic transmission, neurotransmitter release and other neuronal activity), nitrogen metabolism (e.g., nitric oxide biosynthesis, nitrogen utilization and other nitrogen metabolism), non-vertebrate processes (non-verterburate processes), nucleoside, nucleotide and nucleic acid metabolism (e.g., DNA replication, DNA degradation, DNA recombination, DNA repair, chromatin packaging and remodeling, cyclic nucleotide metabolism, nucleoside-verterburate metabolism, Transport of nucleotides and nucleic acids, other nucleosides, metabolism of nucleotides and nucleic acids, purine metabolism, pyrimidine metabolism, RNA catabolism, RNA localization, regulation of nucleosides, nucleotide metabolism, reverse transcription, RNA metabolism, tRNA metabolism, mRNA capping, mRNA terminal processing and stability, mRNA polyadenylation, mRNA splicing, general mRNA transcriptional activity, other mRNA transcription, mRNA transcriptional extension, mRNA transcriptional initiation, mRNA transcriptional regulation, and mRNA transcriptional termination), tumorigenesis (e.g., oncogenes, tumor suppressor genes, and other tumorigenesis-associated processes), phosphate metabolism (e.g., phosphate transport, polyphosphate biosynthesis, polyphosphate catabolism, phosphate metabolism, and other regulation of phosphate metabolism), protein metabolism and modification (e.g., proteolysis, amino acid activation and other protein metabolism complexes, protein biosynthesis, protein assembly, protein expression, protein folding, translation regulation, protein ADP-ribosylation, protein acetylation, protein disulfide isomerase response, protein glycosylation, protein methylation, protein phosphorylation, protein-lipid modification), protein targeting and localization (e.g., asymmetric protein targeting and other protein targeting and localization), sensory perception (e.g., olfactory, taste, auditory, pain sensation, pheromone response, visual and other sensory perception), sulfur metabolism (e.g., sulfur redox metabolism and other sulfur metabolism), cellular communication (e.g., cell adhesion mediated signaling, extracellular matrix protein mediated signaling, ligand mediated signaling, and steroid hormone mediated signaling), cell surface receptor mediated signaling (e.g., cytokine and chemokine mediated signaling, protein acetylation, protein disulfide isomerase response, protein glycosylation, protein methylation, protein phosphorylation, protein-lipid modification, protein targeting and localization), sensory perception (e.g., olfactory, and other sensory perception), sulfur metabolism (e.g., sulfur redox metabolism, G-protein mediated signaling, receptor protein serine/threonine kinase signaling pathways, receptor protein tyrosine kinase signaling pathways, and other receptor mediated signaling pathways), intracellular signaling cascades (e.g., calcium mediated signaling, jak-stat cascade, JNK cascade, MAPKKK cascade, NF-. kappa.B cascade, NO mediated signaling, and other intracellular signaling cascades), and other signaling processes.

The protein and/or RNA components of the various biological pathways disclosed herein and their relationship to each other are known to those of ordinary skill in the art and can be found, for example, in the KEGG pathway database on the Internet (http:// www.genome.jp/KEGG/pathway. html).

In certain embodiments, the cells or cell lines of the invention express at least one functional RNA or protein involved in a biological pathway. In certain embodiments, a cell or cell line of the invention expressing an RNA or protein of interest also expresses at least one functional RNA or protein component of a biological pathway. In certain embodiments, the cells or cell lines of the invention express at least one functional RNA or protein component of a biological pathway, which may or may not be expressed in the same type of cell or cell line that has not been engineered. In certain embodiments, the cells or cell lines of the invention express at least two functional RNA or protein components of a biological pathway sufficient to confer at least one activity of the biological pathway in the cell or cell line, also referred to herein as "expression of a functional biological pathway". In certain embodiments, expression of a biological pathway may include expression of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 components of the biological pathway. In certain embodiments, expression of a biological pathway may include expression of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or all of the components of the biological pathway. Wherein expression of at least one functional RNA or protein component of a biological pathway in a cell or cell line in which the biological pathway does not naturally occur results in reconstitution of at least one activity of a functional biological pathway in the cell or cell line. Wherein expression of at least one functional RNA or protein component of a biological pathway in a cell or cell line in which the biological pathway naturally occurs results in an increase in at least one activity of a functional biological pathway in the cell or cell line. Wherein expression of at least one functional RNA or protein component of a biological pathway in a cell or cell line in which the biological pathway naturally occurs can result in alteration of the network or overall activity of the pathway in the cell. In certain embodiments, the protein of interest expressed in the cells or cell lines of the invention may be modified, post-translationally modified, glycosylated, or altered by co-expression of at least one RNA or protein component of a biological pathway. In certain embodiments, the protein of interest is an IgG or antibody and the pathway is a glycosylation pathway. In certain embodiments, the cell lines in the subject panel of cell lines that each express the same protein of interest (e.g., antibody) express at least one RNA or protein component of a biological pathway (e.g., a glycosylation pathway). In certain embodiments, at least one functional RNA or protein component of a biological pathway can interact with (e.g., transiently or chronically modify, alter, glycosylate, or bind to) a protein of interest expressed in a cell. However, protein-protein interactions between the expressed functional proteins are not necessary. For example, the cells and cell lines of the invention may express two or more functional proteins that are related to each other by being components of the same biological pathway, although the two or more functional proteins may not directly interact with each other. The biological pathway may or may not naturally occur in the cell or cell line of the invention expressing two or more functional protein components of the biological pathway. In the first case, expression of two or more functional protein components of a biological pathway may result in an increase in at least one activity of the biological pathway in a cell or cell line. In the latter case, expression of two or more functional protein components of a biological pathway may result in the reconstitution of at least one activity of the entire biological pathway in a cell or cell line. In certain embodiments, at least one or more components of a biological pathway may be naturally expressed by a cell or cell line.

In certain embodiments, the present invention provides a cell or cell line stably expressing at least one functional protein involved in a biological pathway of interest, wherein the cell or cell line is cultured in the absence of selective pressure and wherein the cell or cell line consistently expresses the at least one functional protein as described herein. Examples of biological pathways that may be used according to the present invention include, but are not limited to, those involved in unfolded protein response ("UPR"); cell growth, cell survival, cell death, cell health; protein expression, production, folding, secretion, membrane integration, modification or post-translational modification (including glycosylation or enzymatic modification); pathways that are enriched for or more highly expressed or active in antibody producing cells, mammalian glandular cells, salivary cells, or cells that highly express engineered proteins compared to other cell types.

Any of the cells described herein can be used as host cells for expressing functional RNAs or proteins involved in biological pathways. Examples of cells that can be used to express at least one functional RNA or protein involved in a biological pathway include, but are not limited to: CHO, CHOK1, CHOKiSV, PerC6, NS0, 293T and insect cells. In certain embodiments, the CHO cell may be used to express at least one component of the UPR pathway. In certain embodiments, the CHO cell may be used to express at least one component of the glycosylation pathway. In some other embodiments, such cells may also express a protein of interest (e.g., an antibody) that is glycosylated.

In certain embodiments, the host cell does not express any component of the pathway prior to introducing into the host cell a nucleic acid encoding or activating transcription of a functional protein of interest involved in the biological pathway. In other embodiments, the host cell expresses at least 1, 2, 5, 10, 15, 20, or at least 25 components of the pathway prior to introducing into the host cell a nucleic acid encoding or activating transcription of a functional protein of interest involved in the biological pathway.

In certain embodiments, in addition to expressing one or more functional protein components of a biological pathway, the cells and cell lines of the invention express one or more functional proteins that modulate the biological pathway and/or at least one component thereof, for example, by affecting the expression or function of one or more functional protein components in the biological pathway. Such effects may include, but are not limited to: post-translational modification (e.g., glycosylation), yield, folding, assembly, and/or secretion of one or more functional proteins in a biological pathway. Examples of genes or RNAs (including mutated, spliced, and processed forms) that may be involved in biological pathways (e.g., involved in UPR, cell survival, protein production, folding, assembly, modification, glycosylation, proteolysis, secretion, integration into the cell's membrane, cell surface presentation, or a combination of such pathways) and expression products encoded by such genes or RNAs include, but are not limited to: spliced ATF6a, IRE1a, IRE1b, PERCDC, ATF4, YYI, NF-YA, NF-YB, NF-YC, spliced XBP1 and EDEM1(UPR gene); NRF2, HERP, XIAP, GADD34, PPI a, b, and g, and DNAJC3 (off gene); spliced blip-1 and XBP1 (genes expressed in B cells); CRT (CaBP3), CNX, ERp57(PDIA3), BiP, BAP, ERdj3, CaBP1, GRP94(CaBP4), ERp72(PDIA4) and cyclophilin B (folding/secreting gene-class I chaperone); BiP, BAP, ERdj3, CaBP1, GRP94(CaBP4), ERp72(PDIA4) and cyclophilin B (class 2 chaperones); SDF2-L (glycosylation gene); ERO1a and b, ERAD, mannosidase 1, HRD1 (oxidative gene); STC1 and 2, SERCA1 and 2, COD1 (calcium pump); INO1, SREBP1DC, SREBP2DC, and PYC (adipogenic/metabolic gene); sec61Pa, b and g (transport/membrane integration gene); and Bcl-2sp, Bcl-xL, Bim, Ku70, VDAC2, BAP31 and 4-3-3 (cell survival/anti-apoptosis genes).

In certain embodiments, the cells and cell lines of the invention express at least a second protein of interest in addition to the expression of the first protein of interest or functional biological pathway or one or more components thereof, wherein the expression of the second protein of interest is affected or altered by the expression of the first protein of interest or functional biological pathway or one or more components thereof. Examples of such effects include, but are not limited to, effects at the mRNA transcription, splicing, trafficking, protein translation, post-translational modifications (e.g., glycosylation) and protein trafficking, folding, assembly, membrane integration, secretion and overall yield (yield) levels. For example, expression of a first protein of interest or functional biological pathway or one or more components thereof can result in increased or more efficient or correct mRNA transcription, splicing, transport, protein translation, post-translational modifications (e.g., glycosylation) and protein trafficking, folding, assembly, membrane integration, secretion and/or overall yield (yield) of a second protein of interest. In certain embodiments, the second protein of interest is a biological product. Examples of biologicals are provided further below. In certain embodiments, the cells or cell lines of the invention co-express an antibody and a glycosylation pathway.

In certain embodiments, in particular, a protein expressed by a cell or cell line used in a method is a protein that has not been previously obtained in a cell of a cell type (which does not normally express the protein without cell or genetic modification) with respect to its functional cell line. Without being bound by theory, it is believed that some reasons for which such cell lines are still not possible to date include: proteins are highly complex or only expressed in specialized or rare cells without cellular or genetic engineering and if cells expressing the protein are not made in large quantities, it is not possible to identify one of the possibly rare engineered cells in which the protein can be properly expressed, assembled, modified, localized, functionally conferred, associated with a cofactor or not associated with cytotoxicity; or because there are no known protein ligands or modulators for identifying cells or cell lines that express the functional form of the protein; or because the protein is cytotoxic when expressed outside its natural background, e.g., in a background in which it is not naturally expressed.

Cells and cell lines of the invention may be prepared that consistently express any protein of interest, including but not limited to proteins located in the cytoplasm, proteins that integrate or bind to at least one membrane of the cell, cell surface localized or secreted proteins, or any combination of such proteins. Such proteins include heteromultimeric ion channels, ligand-gated (e.g., GABA a receptors), ion channels (e.g., CFTR), heteromultimeric ion channels, voltage-gated (e.g., NaV), heteromultimeric ion channels, non-ligand-gated (epithelial sodium channels, ENaC), heterodimeric GPCRs (e.g., opioid receptors, taste receptors, including sweet, uman and bitter receptors), other GPCRs, orphan GPCRs, GCC, opioid receptors, growth hormone receptors, estrogen/hgh, nuclear or membrane-bound TGF receptors, PPAR nuclear hormone receptors, nicotine/Ach and immune receptors such as B cell/T cell receptors, chemosensory receptors such as acid, cold, warm, heat, fat, fatty acid or lipid taste or sensation, milk taste, touch, pain, mouth feel, and tingling sensation.

The cells and cell lines of the invention can express functional proteins, including any of the proteins or combinations of proteins listed in tables 7-22 (mammalian G proteins, human orphan GPCRs, human opioid receptors, human olfactory receptors, canine olfactory receptors, mosquito olfactory receptors, other heteromultimeric receptors, and GABA receptors).

The cells and cell lines of the invention have a number of attributes that make them particularly advantageous for any use where it is desired that the cells consistently express a functional protein of interest over time. The terms "stable" or "consistent" when applied to the expression of a protein and the function of a protein are intended to distinguish the cells and cell lines of the invention from cells having transient expression or variable function, as the terms "stably expressed" and "transiently expressed" will be understood by those of ordinary skill in the art. The cells or cell lines of the invention may have stable or consistent expression of a functional protein with less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20% change for at least 2-4 days.

In certain embodiments, the stability of the cells or cell lines of the invention can be distinguished from transient expression.

In certain embodiments, the stability of a cell or cell line of the invention can be maintained in the absence of selective pressure against one or more RNA or protein of interest. In certain embodiments, the stability of a cell or cell line of the invention can be maintained at a minimum or reduced level or amount of selection pressure or drug as compared to the level or amount normally used or used immediately after introduction of a nucleic acid encoding an RNA or protein of interest into an engineered cell population or normally used in a method of selecting cells or cell lines having an amplified copy number of the nucleic acid.

In certain embodiments, the level of stability observed in a cell or cell of the invention is higher compared to the normal level that can be achieved in a cell or cell line produced in a general or majority of cells of the same cell type.

In certain embodiments, the level of stability observed in the cell or cells of the invention is characterized by: lower variability in the expression level, activity or function of an RNA or protein of interest compared to normal values that can be achieved in cells or cell lines produced in general or in most cells of the same cell type.

In certain embodiments, the length of the duration of stability of expression of an RNA or protein of interest in a cell or cell line of the invention is longer than normal, which can be achieved in a general or majority of cells of the same cell type.

In certain embodiments, the stability of a cell or cell line of the invention can be maintained with minimal or no observed cytotoxicity associated with expression of an RNA or protein of interest (as compared to values achieved in normal or most cells of the same cell type).

In certain embodiments, the stability of a cell or cell line of the invention can be maintained with minimal or no change in the functional form, function, physiology, pharmacology, assembly, localization, post-translational modification, glycosylation, enzymatic modification, proteolytic modification, or stoichiometry of the RNA or protein of interest in culture over time (as compared to values that can be achieved in normal or most cells of the same type). In certain embodiments, such properties can be determined by characterizing the pharmacological properties or response of a cell or cell line using a compound that modulates the expression or function of an RNA or protein of interest in a cell-based assay.

In various embodiments, the cells or cell lines of the invention express functional RNA or protein of interest, i.e., the cells are functionally identical after at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 days of growth, wherein consistent expression or consistent function means that the expression level does not change by more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% over 2 to 4 days of continuous cell culture; expression levels do not change by more than 1%, 2%, 4%, 6%, 8%, 10% or 12% over 5 to 15 days of continuous cell culture; expression levels do not change by more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18% or 20% over 16 to 20 days of continuous cell culture; expression levels do not change by more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24% over 21 to 30 days of continuous cell culture; expression levels do not change by more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% over 30 to 40 days of continuous cell culture; expression levels do not change by more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% over 41 to 45 days of continuous cell culture; expression levels do not change by more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% over 45 to 50 days of continuous cell culture; expression levels do not change by more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30% or 35% over 45 to 50 days of continuous cell culture; expression levels do not change by more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28% or 30% or 35% over 50 to 55 days of continuous cell culture; expression levels do not change by more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30% or 35% over 50 to 55 days of continuous cell culture; expression levels do not change by more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35% or 40% over 55 to 75 days of continuous cell culture; expression levels do not change by more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% over 75 to 100 days of continuous cell culture; (ii) the expression level does not change by more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% over 101 to 125 days of continuous cell culture; expression levels do not change by more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% over 126 to 150 days of continuous cell culture; expression levels do not change by more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% over 151 to 175 days of continuous cell culture; expression levels do not change by more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% over 176 to 200 days of continuous cell culture; expression levels do not change by more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% over 200 days of continuous cell culture.

In various embodiments, the cells or cell lines of the invention are stable, i.e., the cells or cell lines maintain consistent expression, amount, yield, function, or activity of an RNA or protein of interest for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 days, wherein "consistent" means that the expression level, amount, yield, function, or activation of the RNA or protein of interest changes by no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% over 2 to 4 days of continuous cell culture; no more than 1%, 2%, 4%, 6%, 8%, 10% or 12% change over 5 to 15 days of continuous cell culture; (ii) does not change by more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18% or 20% over 16 to 20 days of continuous cell culture; no more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24% change over 21 to 30 days of continuous cell culture; no more than a 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% change over 30 to 40 days of continuous cell culture; no more than a 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% change over 41 to 45 days of continuous cell culture; no more than a 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% change over 45 to 50 days of continuous cell culture; no more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, or 35% change over 45 to 50 days of continuous cell culture; no more than a 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% or 35% change over 50 to 55 days of continuous cell culture; no more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, or 35% change over 50 to 55 days of continuous cell culture; (ii) does not change by more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35% or 40% over 55 to 75 days of continuous cell culture; (ii) no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% change over 75 to 100 days of continuous cell culture; (ii) does not change by more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% over 101 to 125 days of continuous cell culture; no more than a 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% change over a 126 to 150 day continuous cell culture; no more than a 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% change over 151 to 175 days of continuous cell culture; no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% change upon 176 to 200 days of continuous cell culture; the change is no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% over 200 days of continuous cell culture.

In various embodiments, the cells or cell lines of the invention are stable, i.e., the cells or cell lines maintain consistent expression, amount, yield, function, or activity of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 days of at least two RNAs or proteins of interest, wherein "consistent" means that the expression level, stoichiometry, amount, yield, function, or activity of an RNA or protein of interest does not change by more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% over 2 to 4 days of continuous cell culture; no more than 1%, 2%, 4%, 6%, 8%, 10% or 12% change over 5 to 15 days of continuous cell culture; no more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, or 20% change over 16 to 20 days of continuous cell culture; no more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24% change over 21 to 30 days of continuous cell culture; no more than a 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% change over 30 to 40 days of continuous cell culture; no more than a 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% change over 41 to 45 days of continuous cell culture; no more than a 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% change over 45 to 50 days of continuous cell culture; no more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, or 35% change over 45 to 50 days of continuous cell culture; no more than a 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% or 35% change over 50 to 55 days of continuous cell culture; no more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, or 35% change over 50 to 55 days of continuous cell culture; (ii) does not change by more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% over 55 to 75 days of continuous cell culture; (ii) no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% change over 75 to 100 days of continuous cell culture; (ii) does not change by more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% over 101 to 125 days of continuous cell culture; no more than a 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% change over a 126 to 150 day continuous cell culture; no more than a 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% change over 151 to 175 days of continuous cell culture; no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% change upon 176 to 200 days of continuous cell culture; the change is no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% over 200 days of continuous cell culture.

Cells having the desired properties and stably expressing the functional protein can be selected. Any desired property that can be detected can be selected. Such features will be known to those of ordinary skill in the art. Such properties include, as non-limiting examples:

fragility, morphology and attachment to solid surfaces, monodispersity by trypsin or cell dissociation reagents, adaptability to automated culture conditions, performance under serum-containing conditions, performance under serum-free conditions, convertibility to serum-free suspension conditions, propensity to form clots, propensity to form monodisperse cell layers after passaging, restorative forces, propensity to remain attached to growth chamber surfaces under different force flow addition steps, unbroken nuclei, lack of intracellular vacuoles, lack of microbial contamination, lack of mycoplasma, lack of viral contamination, clonogenic capacity, consistency of macroscopic physical properties of cells within pores, propensity to grow below/above room temperature, propensity to tolerate various temperatures over various time periods, propensity for balanced cellular uptake of plasmids/oligonucleotides/fluorescent probes/peptides/proteins/compounds Cells are subject to incubation with DMSO/EtOH/MeOH, organic solvents/detergents, cells are subject to UPR induced maintenance, cells are subject to exposure to DTT, cells are subject to infection by virus/lentivirus/cosmid vectors, endogenous expression of desired RNA/protein or lack thereof, chromosome number, chromosomal abnormalities, compliance with growth at 5/6/7/8/9pH, tolerance to UV/mutagens/radiation, ability to maintain the above features under altered/artificial/scaled up growth conditions (i.e. including reactor).

The cells and cell lines of the invention have enhanced properties compared to cells and cell lines produced by conventional methods. For example, the cells and cell lines of the invention have enhanced expression stability and/or expression levels even when maintained in culture without selective pressure (including, e.g., antibiotics or other drugs). In other embodiments, the cells and cell lines of the invention have high Z' values in various assays. In still other embodiments, the cells and cell lines of the invention are improved in the context of their expression of physiologically relevant protein activity as compared to more conventionally engineered cells. These properties enhance and improve the ability of the cells and cell lines of the invention to be used for any purpose, whether in assays to identify modulators, for cell therapy, for protein production or any other use, and improve the functional attributes of the identified modulators.

In certain embodiments, a further advantageous property of the cells and cell lines of the invention is that they stably express a protein of interest in the absence or reduction of drugs or other selection pressures that are commonly used or that can be used immediately after introduction of a nucleic acid encoding the protein of interest into the cells or that can be used in methods of selecting cells having amplified copy numbers of the nucleic acid. Without being bound by theory, cytotoxicity associated with expression of an RNA or protein of interest in a cell of a cell type that does not normally express the RNA or protein of interest may reflect: conditions sufficient for non-cytotoxic expression of the RNA or protein of interest are not obtained close to or reproduced in the engineered cells. In certain embodiments, engineered cells with reduced or absent cytotoxicity associated with expression of such an RNA or protein of interest are preferred, as reduced or absent cytotoxicity may indicate that improved conditions for expression or function of the RNA or protein of interest have been achieved. In certain embodiments, the engineered cell or cell line may express an RNA or protein of interest that is not naturally expressed or is not naturally expressed in the absence of the cell or genetic modification, in the same cell type, in the absence of relevant cytotoxicity. In certain embodiments, the engineered cell or cell line may express an RNA or protein of interest that is not naturally functional or correctly expressed, folded, assembled, modified, post-translationally modified, localized, or inactive in the same type of cell in the absence of the cell or genetic modification. In certain embodiments, the methods of the invention result in a cell or cell line comprising functional, stable, viable or non-cytotoxic expression of an RNA or protein of interest previously thought to be cytotoxic when expressed in a general or majority of cells of the same cell type. Thus, in a preferred embodiment, the cells and cell lines of the invention are maintained in culture without any selective pressure. In other embodiments, the cells and cell lines are maintained in the absence of any drugs or antibiotics. As used herein, cell maintenance refers to culturing cells after they have been selected as described for protein expression. Maintenance does not refer to an optional step of growing the cells under selective pressure (e.g., antibiotics) prior to cell sorting, where the introduced intracellular marker allows enrichment of stable transfectants in the mixed population.

Drug-free and selective pressure-free cell maintenance of the cells and cell lines of the invention provides a number of advantages. For example, drug resistant cells may not express the transgene of interest co-transfected at sufficient levels, as selection depends on the survival of cells that have taken up the drug resistant gene and may or may not contain the transgene. In addition, the need for cytotoxicity or drug selection or other selection pressure to maintain expression or function of the RNA or protein of interest indicates that the optimal conditions required for expression or function of the RNA or protein of interest are not achieved. In addition, selection drugs and other selection pressures are often mutagenic or otherwise interfere with the physiology of the cell, leading to biased results in cell-based assays. For example, selection of drugs can reduce susceptibility to apoptosis (Robinson et al, Biochemistry, 36 (37): 11169-11178(1997)), increase DNA repair and drug metabolism (Deffie et al, Cancer Res.48 (13): 3595-3602(1988)), increase cell pH (Thiebaut et al, J Histochem Cytomem.38 (5): 685 690 (1990)), Roepe et al, biochemistry.32 (41): 11042-11056(1993)), Simon et al, Proc Natl Acad Sci USA.91 (3): 1128-1132(1994)), decrease lysosomal and endosomal pH (Schinder et al, biochemistry.35 (9): 2811-2817 (1996)), Altan et al, J exp.Med.187 (10): 1583) (1998), decrease in plasma membrane potential (11032-11041) (Biochem et al, 11041-11041 (11041) (biochem et al, 11023, 11032-biochem et al, 11041) (biochem et al, 11032), proc Natl Acad SciSciUSA.90 (1): 312-: 4432-4437(1999)). Thus, the cells and cell lines of the invention allow screening assays free of artifacts caused by selection pressure. In certain preferred embodiments, the cells and cell lines of the invention are not cultured with a selection pressure factor, such as an antibiotic, either before or after cell sorting, such that cells and cell lines having the desired properties are isolated by sorting even when not starting from an enriched cell population.

The cells and cell lines of the invention have enhanced stability in the context of expression and expression levels (RNA or protein) compared to cells and cell lines produced by conventional methods. To identify cells and cell lines with such stable expression profiles, the expression of the protein of interest in the cells or cell lines is measured over a time course and the expression levels are compared. Stable cell lines will continue to express (RNA or protein) throughout this time course. In certain aspects of the invention, the time course may be at least 1 week, 2 weeks, 3 weeks, etc., or at least 1 month, or at least 2, 3, 4, 5, 6, 7, 8, or 9 months, or any length of time therebetween.

Isolated cells and cell lines can be further characterized, for example, by PCR, RT-PCR, qRT-PCR, and single endpoint RT-PCR to determine the absolute and relative amounts of expressed (RNA) (in the case of multi-subunit proteins or multiple proteins of interest). Preferably, the amplification level of the subunits of the multi-subunit protein is substantially the same in the cells and cell lines of the invention.

In other embodiments, the expression of a functional protein of interest is determined over time. In these embodiments, stable expression is measured by comparing the results of the functional assay over time. Assays of cells and cell lines based on functional assays provide the benefit of identifying cells and cell lines that not only stably express proteins (RNA or proteins), but also stably produce and appropriately process (e.g., post-translational modifications, subunit assembly, and localization within the cell) proteins to produce functional proteins.

The cells and cell lines of the invention have the further advantageous property of providing assays with high reproducibility, as evidenced by their Z' factor. See Zhang JH, Chung TD, OldenburgKR, "a Simple statistical parameter for Use in evaluation and Validationof High through screen analysis assays," j.biomol. 4(2): 67-73, which are incorporated herein by reference in their entirety. The Z' value relates to the quality of the cell or cell line as it reflects the extent to which the cell or cell line will respond consistently to the modulator. Z' is a statistical calculation that takes into account the range of signal-to-noise ratios and signal variability (i.e., from well to well) across the functional response of the multi-well plate to the reference compound. Z' was calculated using data from multiple wells with positive controls and multiple wells with negative controls. The ratio of the standard deviations of their combinations was multiplied by 3 to the difference factor according to the following equation, and the average was subtracted from 1 to give Z'

Factor Z' 1- ((3. delta.)_{Positive control}+3δ_{Negative control})/(μ_{Positive control}-μ_{Negative control}))

If the factor is 1.0, this would indicate an ideal assay with a theoretical maximum of Z', no variability and infinite dynamic range. As used herein, "high Z '" refers to a Z' factor having any fractional number between at least 0.6, at least 0.7, at least 0.75, or at least 0.8, or 0.6 to 1.0. In the case of complex targets, high Z 'means a Z' of at least 0.4 or greater. A score close to 0 is undesirable because it indicates that there is overlap between the positive and negative controls. In the industry, for simple cell-based assays, Z ' scores of up to 0.3 are considered marginal scores, Z ' scores between 0.3 and 0.5 are considered acceptable, and Z ' scores above 0.5 are considered excellent. Cell-free or biochemical assays can approach scores for cell-based systems, which tend to be lower because of higher Z 'scores, but Z' cell-based systems are complex.

As will be appreciated by those of ordinary skill in the art, cell-based assays using conventional cells expressing even single-chain proteins typically do not achieve a Z' of greater than 0.5 to 0.6. Even if reported in the art, cells with engineered expression of multi-subunit proteins (from introduced coding sequences or gene activation) would be lower due to their added complexity. Such cells are not reliable for use in assays, as the results will not be reproducible. On the other hand, the cells and cell lines of the invention have higher Z' values and advantageously produce consistent results in the assay. In fact, the cells and cell lines of the present invention provide the basis for High Throughput Screening (HTS) compatible assays, as they generally have different values than conventionally produced cells. In certain aspects of the invention, the cells and cell lines result in a Z' of at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, or at least 0.8. For the cells and cell lines of the invention, even Z' values of at least 0.3-0.4 are advantageous, since the protein of interest is a multigene target. In other aspects of the invention, the cells and cell lines of the invention result in a Z' of at least 0.7, at least 0.75, or at least 0.8, even after the cells have been maintained for multiple passages, e.g., between 5-20 passages (including any integer between 5 and 20). In certain aspects of the invention, the cells and cell lines result in a Z' of at least 0.7, at least 0.75, or at least 0.8 in cells and cell lines maintained for 1, 2, 3, 4, or 5 weeks or 2, 3, 4, 5, 6, 7, 8, or 9 months, including any time period therebetween.

In certain embodiments, the cells and cell lines of the invention express a protein of interest, wherein one or more physiological properties remain substantially unchanged over time.

The physiological characteristic includes any observable, detectable or measurable property of the cell or cell line other than expression of the protein of interest.

In certain embodiments, expression of the protein of interest may alter one or more physiological properties. The alteration of the physiological property includes any change in the physiological property due to expression of the protein of interest, for example, stimulation, activation or enhancement of the physiological property, or inhibition, blocking or attenuation of the physiological property. Without being bound by theory, in such embodiments, the one or more consistent physiological properties indicate that the functional expression of the protein of interest also remains consistent. In particular embodiments, one or more consistent physical properties associated with taste receptors (e.g., sweet, umami, or bitter taste receptors), discussed in more detail below, may be used to monitor the expression of functional taste receptors.

Without being bound by theory, the present invention provides a method of culturing a plurality of cells or cell lines expressing a protein of interest under consistent culture conditions, wherein cells or cell lines can be selected that have one or more desired properties (e.g., stable expression) and/or one or more substantially consistent physiological characteristics of the protein of interest.

In some embodiments, where the physiological property can be measured, the physiological property is determined as an average of the physiological property measured in a plurality of cells or a plurality of cells of a cell line. In certain specific embodiments, the physiological properties of at least 10, 100, 1,000, 10,000, 100,000, 1,000,000, or at least 10,000,000 cells are measured and the average values remain substantially consistent over time.

In certain embodiments, the average value of the physiological property is determined by measuring the physiological property of a plurality of cells or a plurality of cells of a cell line, wherein the cells are in different time periods of the cell cycle. In other embodiments, the cells are synchronized on the cell cycle.

In certain embodiments, the physiological property is observed, detected, measured, or monitored at the single cell level. In certain embodiments, the physiological properties remain substantially consistent over time at the single cell level.

In certain embodiments, the physiological property remains substantially consistent over time if the physiological property does not change by more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or more than 50% over a 12 hour period. In certain embodiments, the physiological property remains substantially consistent over time if the physiological property does not change more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or more than 50% over a 1 day period. In certain embodiments, the physiological property remains substantially consistent over time if the physiological property does not change by more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or more than 50% over a 2 day period. In certain embodiments, the physiological property remains substantially consistent over time if the physiological property does not change by more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or more than 50% over a 5 day period. In certain embodiments, the physiological property remains substantially consistent over time if the physiological property does not change more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or more than 50% over a 10 day period. In certain embodiments, the physiological property remains substantially consistent over time if the physiological property does not change more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or more than 50% over a 20 day period. In certain embodiments, the physiological property remains substantially consistent over time if the physiological property does not change more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or more than 50% over a 30 day period. In certain embodiments, the physiological property remains substantially consistent over time if the physiological property does not change more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or more than 50% over a 40 day period. In certain embodiments, the physiological property remains substantially consistent over time if the physiological property does not change more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or more than 50% over a 50 day period. In certain embodiments, the physiological property remains substantially consistent over time if the physiological property does not change by more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or more than 50% over a 60 day period. In certain embodiments, the physiological property remains substantially consistent over time if the physiological property does not change more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or more than 50% over a 70 day period. In certain embodiments, the physiological property remains substantially consistent over time if the physiological property does not change more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or more than 50% over a period of 80 days. In certain embodiments, the physiological property remains substantially consistent over time if the physiological property does not change more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or more than 50% over a 90 day period. In certain embodiments, the physiological property remains substantially consistent over a period of time if the physiological property does not change more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or more than 50% over the course of passage 1, passage 2, passage 3, passage 5, passage 10, passage 25, passage 50, or passage 100.

Examples of cellular physiological properties include, but are not limited to: growth rate, size, shape, morphology, volume; DNA, RNA, protein, lipid, ion, carbohydrate, or water; expression or amount of endogenous, engineered, introduced, gene-activated, or all genes, RNAs, or proteins; propensity or adaptability to growth under attachment, suspension, serum-containing, serum-free, animal component-free, shaking, resting, or bioreactor growth conditions; propensity or adaptation to grow in or on a chip, array, microarray, slide, plate, multi-well plate, high density multi-well plate, culture flask, roller bottle, bag or jar; propensity or adaptability to growth using manual or automated or robotic cell culture methods; an abundance, level, quantity, amount, or composition of an organelle, compartment, or membrane of at least one cell, including but not limited to cytoplasm, nucleolus, nucleus, ribosome, rough endoplasmic reticulum, golgi apparatus, cytoskeleton, smooth endoplasmic reticulum, mitochondria, vacuole, cytosol, lysosome, centromere, chloroplast, cell membrane, plasma membrane, nuclear envelope, vesicle (e.g., secretory vesicle), or at least one membrane of an organelle; has obtained or has the ability or propensity to obtain at least one function or gene expression signature (of one or more genes) shared by one or more specific cell types or differentiated, undifferentiated or dedifferentiated cell types, including but not limited to: stem cells, pluripotent cells, totipotent cells or specialized cells or tissue-specific cells, including: liver, lung, skin, muscle (including but not limited to: cardiac muscle, skeletal muscle, striated body muscle), pancreas, brain, testis, ovary, blood, immune system, nervous system, bone, cardiovascular system, central nervous system, gastrointestinal tract, stomach, thyroid, tongue, gallbladder, kidney, nose, eye, nail, hair, taste bud or taste cells, neurons, skin cells, pancreatic cells, blood cells, immune cells, red blood cells, white blood cells, killer T cells, endocrine cells, secretory cells, kidney cells, epithelial cells, endothelial cells, human, animal or plant cells; the ability or property to take up natural or synthetic chemicals or molecules, including but not limited to nucleic acids, RNA, DNA, proteins, small molecules, probes, dyes, oligonucleotides (including modified oligonucleotides), or fluorescent oligonucleotides; resistance or ability to the negative or deleterious effects of chemicals or substances that negatively affect cell growth, function or viability, including but not limited to: resistance to infection, drugs, chemicals, pathogens, detergents, UV, adverse conditions, cold, heat, extreme temperatures, agitation, perturbation, vortexing, lack or low levels of oxygen, lack or low levels of nutrients, toxins, venoms, viruses, or compounds, treatments, or agents that have an adverse effect on cells or cell growth; for in vitro testing, cell-based assays, biochemical or biological testing, implantation, cell therapy, or secondary assays, including but not limited to: large scale cell culture, miniaturized cell culture, automated cell culture, robotic cell culture, standardized cell culture, drug discovery, high throughput screening, cell-based assays, functional cell-based assays (including but not limited to membrane potential assays, calcium flux assays, reporter assays, G protein receptor assays), ELISA, in vitro assays, in vivo applications, secondary assays, compound testing, binding assays, panning assays, antibody panning assays, phage display, imaging studies, microscopic imaging assays, immunofluorescence studies, RNA, DNA, protein or biologic production or purification, vaccine development, cell therapy, implantation into an organism, animal, human, or plant, isolation of factors secreted by cells, preparation of cDNA libraries, or infection by pathogens, viruses, or other agents; and other observable, measurable or detectable physiological characteristics such as: biosynthesis of at least one metabolite, lipid, DNA, RNA, or protein; chromosome silencing, activation, heterolysis, autosomal (euchromosis) or recombination; gene expression, gene silencing, gene splicing, gene recombination, or gene activation; RNA production, expression, transcription, processing, splicing, transport, localization, or modification; protein production, expression, secretion, folding, assembly, transport, localization, cell surface presentation, secretion or integration into the membrane of a cell or organelle; protein modifications include, but are not limited to, post-translational modifications, processing, enzymatic modifications, proteolysis, glycosylation, phosphorylation, dephosphorylation; cell division, including mitosis, meiosis or division or cell fusion; high level of RNA or protein production or yield.

The physiological properties can be observed, detected, or measured using conventional assays known in the art, including, but not limited to, reference guidelines and manuals such as the tests and methods described in Current Protocols series. This system includes common protocols in different fields and is available through Wiley Publishing House. Protocols in such reference guidelines illustrate methods that can be used to observe, detect, or measure physiological properties of cells. One of ordinary skill in the art can readily recognize that any one or more of such methods can be used to observe, detect, or measure the physiological properties disclosed herein.

Many markers, dyes or reporter molecules (including protein markers expressed as fusion proteins comprising an autofluorescent protein) that can be used to measure the level, activity or content of cellular compartments or organelles including but not limited to ribosomes, mitochondria, ER, rER, golgi apparatus, TGN, vesicles, endosomes and plasma membranes in a cell are compatible with testing of single living cells. In certain embodiments, fluorescence activated cell sorting or cell sorter may be used. In certain embodiments, the isolated or produced cell or cell line comprising the RNA or protein of interest may be tested using such labels, dyes, or reporter molecules simultaneously with, after, or prior to isolation, testing, or production of the cell or cell line comprising the RNA or protein of interest. In certain embodiments, the level, activity or content of one or more cellular compartments or organelles can be correlated with increased, native, non-cytotoxic, viable or optimal expression, function, activity, folding, assembly modification, post-translational modification, secretion, cell surface presentation, membrane integration, pharmacology, productivity, or physiology of an RNA or protein of interest. In certain embodiments, a cell or cell line can be isolated that comprises a level, activity, or content of at least one cellular compartment or organelle associated with increased, native, non-cytotoxic, viable, or optimal expression, function, activity, folding, assembly modification, post-translational modification, secretion, cell surface presentation, membrane integration, pharmacology, productivity, or physiology of an RNA or protein of interest. In certain embodiments, a cell or cell line can be isolated comprising an RNA or protein of interest and a level, activity, or content of at least one cellular compartment or organelle associated with increased, native, non-cytotoxic, viable, or optimal expression, function, activity, folding, assembly modification, post-translational modification, secretion, cell surface presentation, membrane integration, pharmacology, productivity, or physiology of the RNA or protein of interest. In certain embodiments, the separation of cells is performed using cell sorting or fluorescence activated cell sorting.

In certain embodiments, a population of different cells that can be engineered to comprise an RNA or protein of interest can be contacted with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, or 200 additional nucleic acid sequences, introduced into the population of cells, or engineered to comprise the nucleic acid sequences, simultaneously, prior to, or after isolation, testing, or generation of the cell or cell line engineered to comprise the RNA or protein of interest. In certain embodiments, the additional nucleic acid may be selected from the group consisting of: an RNA, DNA, or gene encoding a modulator of Unfolded Protein Response (UPR); an RNA, DNA, or gene that is modulated in a UPR or in the status of a UPR; RNA, DNA, or genes that regulate cell growth, cell survival, cell death, cell health; RNA, DNA, or gene that modulates expression, production, folding, secretion, yield, membrane integration, modification, or post-translational modification (including glycosylation or enzymatic modification) of RNA or protein; RNA, DNA, or genes enriched in antibody-producing cells compared to other cell types.

In certain embodiments, expression of at least one nucleic acid in a cell may be associated with increased, native, non-cytotoxic, viable or optimal expression, function, activity, folding, assembly modification, post-translational modification, secretion, cell surface presentation, membrane integration, pharmacology, productivity, or physiology of an RNA or protein of interest. In certain embodiments, a cell or cell line can be isolated that comprises at least one nucleic acid associated with increased, native, non-cytotoxic, viable or optimal expression, function, activity, folding, assembly modification, post-translational modification, secretion, cell surface presentation, membrane integration, pharmacology, productivity, or physiology of an RNA or protein of interest. In certain embodiments, a population of different cells that may be engineered to comprise an RNA or protein of interest is contacted with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, or 200 additional nucleic acid sequences, either simultaneously with, prior to, or after isolation, testing, or generation of a cell or cell line engineered to comprise the RNA or protein of interest, introduced into or engineered to comprise the nucleic acid sequence, and the cell or cell line may be isolated, comprising the RNA or protein of interest and the enhanced, increased, native, non-cytotoxic, or non-cytotoxic RNA or protein of interest, Viable or optimal expression, function, activity, folding, assembly modification, post-translational modification, secretion, cell surface presentation, membrane integration, level, activity or content of at least one cellular compartment or organelle of pharmacological, productive or physiological relevance. In certain embodiments, the separation of cells is performed using cell sorting or fluorescence activated cell sorting.

The cells or preparations made from the cells can be tested using methods and assays (including sequencing, PCR methods (including PCR, RT-PCR, qRT-PCR, RACE), hybridization methods (including northern and southern blotting), FISH, in situ hybridization, microscopy (including fluorescence, electron, confocal or immunofluorescence microscopy) or array or microarray assays such as gene chips or protein arrays) to analyze the content, organization, expression or profile of DNA, RNA, protein, for example to identify expression profiles of one or more genes whose expression affects, is beneficial or detrimental to the target (e.g., by affecting its functionality, viability or stable expression) or physiological properties of the cells. In certain embodiments, such tests are performed to identify one or more endogenous factors that affect, benefit or compromise functional, viable or stable expression of a protein of interest or physiological property of interest.

Cells or preparations made from cells can be tested using methods and tests (including centrifugation, ultracentrifugation, floating, sucrose gradients, HPLC, FPLC, subcellular fractionation, metabolite analysis, chemical composition analysis, chromosome dispersion, DAPI labeling, NMR, enzyme assays, ELISA) to analyze or characterize cell contents (including metabolites, DNA, RNA, proteins, membranes, lipids, carbohydrates, or organelles);

Proteins produced by cells can be tested using sequencing, antibody binding ELISA, or activity assays to assess their sequence, function, form, folding, membrane integration, abundance, yield, post-translational modification, glycosylation, phosphorylation, cleavage, proteolysis, or degradation.

Cells can be tested and characterized by testing cell growth, mitosis, meiosis, gene integration, gene activation, gene introduction, or gene expression or silencing. In certain embodiments, such tests are performed to identify the location of integration of any transgene in the genome of the cell.

In certain embodiments, the expression profile (e.g., profile of gene expression or protein expression) of a cell or cell line according to the invention can be compared to the expression profile of a reference cell or cell line. Any method known to those of ordinary skill in the art may be used to measure the expression profile of one or more nucleic acid or amino acid sequences. Exemplary methods are gene chips, protein chips, and the like. In certain embodiments, at least 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the genes in the genome are assayed for expression. In certain embodiments, at least 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the nucleic acid or amino acid sequence in the cell is determined. In certain embodiments, the expression of at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 or more than 100 genes in a genome is determined. In certain embodiments, at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 or more than 100 nucleic acids or amino acid sequences are determined in a cell.

In certain embodiments, the reference cell or cell line is a host cell from which the cell or cell line according to the invention is produced. In other embodiments, the cell or cell line and the reference cell or cell line are derived from the same parental clone. In certain embodiments, the cell or cell line and the reference cell or cell line are derived from the same parent cell. In other embodiments, the reference cell or cell line is a cell or cell line of the cell type to which the cell or cell line according to the invention is designed to be in proximity. Such cell types include, but are not limited to: epidermal keratinocytes (differentiated epidermal cells), epidermal basal cells (stem cells), keratinocytes of nail and toenail, nail bed basal cells (stem cells), hair stem medullary cells (middle hair follicle cells), cortical hair stem cells (clinical hair follicle cells), epidermal hair root sheath cells (clinical hair follicle cells), hair root sheath cells of the henle layer, external hair root sheath cells, hair matrix cells (stem cells), cornea, tongue, oral cavity, esophagus, anal canal, superficial epithelial cells of the stratified squamous epithelium of the distal urethra and vagina, cornea, tongue, oral cavity, esophagus, anal canal, basal cells of the epithelial cells of the distal urethra and vagina (stem cells), urinary tract cells (internal urinary bladder and epithelial cells), salivary gland protease-rich keratinocytes (stem cells), epithelial cells of the mucous glands, salivary gland, urinary tract and salivary gland, Salivary gland serous cells (glycoprotein-rich secretion), von Ebner 'sgland cells (cleansing taste buds) in the tongue, mammary gland cells (milk secretion), lacrimal gland cells (tear secretion), cerumenous gland cells (cerumenous gland cells) in the ear (wax secretion), eccrine sweat gland dark cells (glycoprotein secretion), eccrine sweat gland bright cells (small molecule secretion), apocrine sweat gland cells (fragrance secretion, sex hormone sensitive), moat gland cells (specialized sweat gland) in the eyelid, sebaceous gland cells (lipid-rich sebum secretion), bowman's gland cells (cleansing olfactory epithelium) in the nose, duodenal gland cells (enzyme and alkaline mucus) in the duodenum, seminal vesicle cells (seminal fluid component including fructose required for sperm motility), prostate cells (secretory seminal fluid component), Cells of the bulbourethral gland (mucus secretion), Bartholin gland (secretion of vaginal lubricant), cells of the urinary gland (secretion of mucus), endometrial cells (secretion of carbohydrates), isolated goblet cells of the respiratory and digestive tracts (secretion of mucus), cells of the mucous mucosa (secretion of mucus), cells of the gastric gland enzyme production (secretion of pepsinogen), cells of the gastric gland acid production (secretion of hydrochloric acid), cells of the pancreatic acinus (secretion of bicarbonate and digestive enzymes), cells of the small intestine (secretion of lysozyme), cells of the lung type II (secretion of surfactant), cells of the lung clara, cells of the anterior pituitary, somatrophoblasts, prolactin, thyroid hormone cells, gonadotropic substances, corticotropin cells, intermediate pituitary cells (endocrine cells), secreted melanocyte hormones, large cell neuro secreting cells (secreted oxytocin and/or secreted vasopressin), Intestinal and respiratory cells (serotonin, endorphin, somatostatin, gastrin, secretor, cholecystokinin, insulin, glucagon, and/or bombesin), thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid chief cells, eosinophils, adrenal cells, pheochromocytes, adrenal secreted steroid hormones (mineralocorticoids and glucocorticoids), leydig cells of the testis that secretes testosterone, theca lining cells of the follicle that secretes estrogen, progesterone-secreting disrupted follicle luteal cells (granulosa and membranal luteal cells), juxtaglomerular cells (renin secretion), renal dense macula cells, perirenal pericytes, renal mesangial cells, hepatic cells (hepatocytes), and the like, White adipocytes, brown adipocytes, hepatic adipocytes, glomerular parietal cells, glomerular podocytes, renal proximal tubule brush border cells, loop of medulla thin segment cells, renal distal tubule cells, renal collecting duct cells, type I alveolar cells (lining air space of lung), pancreatic duct cells (cells of the pericardium), striationless duct cells (of sweat glands, salivary glands, mammary glands, etc.) such as main cells and dark cells, duct cells (of seminal vesicles, prostate, etc.), intestinal brush border cells (with microvilli), exocrine gland striatal duct cells, gallbladder epithelial cells, ductal non-ciliated cells, epididymal main cells, epididymal basal cells, vascular and lymphatic vascular endothelial porous cells, vascular and lymphatic vascular endothelial continuous cells, vascular and lymphatic vascular endothelial splenocytes, renal proximal tubule brush border cells, loop of medulla, renal distal tubules, renal collecting duct cells, lung lining air space of lung (lung), pancreatic duct cells (cells of the pericardial sac, salivary glands, mammary glands, etc.) Synovial cells (intra-articular cavity, hyaluronic acid secretion), serosal cells (intra-peritoneum, pleura and pericardial cavity), squamous cells (perilymph space lining the ear), squamous cells (intra-lymph space lining the ear), intra-lymph-sac columnar cells with microvilli (intra-lymph space lining the ear), intra-lymph-sac columnar cells without microvilli (intra-lymph space lining the ear), dark cells (intra-lymph space lining the ear), vestibular membrane cells (intra-lymph space lining the ear), stria vascularis basal cells l (intra-lymph space lining the ear), stria vascularis marginal cells (intra-lymph space lining the ear), claudica cells (intra-lymph space lining the ear), perichester cells (intra-lymph space lining the ear), cerebrospinal fluid cells (cerebrospinal fluid), leptomeningeal squamous epithelial cells, pigmented ciliary epithelium cells of the eye, non-pigmented ciliary epithelium cells of the eye, epithelial cells of the eye, and epithelial cells of the eye, Corneal endothelial cells, respiratory ciliated cells, oviduct ciliated cells (in females), endometrial fibro cells (in females), testicular reticulum ciliated cells l (in males), seminiferous tubule ciliated cells (in males), ciliated ependymal cells of the central nervous system (intracerebroventricular cavity), amelogenic epithelial cells (enamel secretion), semilunar planar epithelial cells of the vestibular apparatus of the ear (proteoglycan secretion), corticoterminal interdental epithelial cells (secretion of a coating covering hair cells), loose connective tissue fibroblasts, corneal fibroblasts (corneal stroma cells), tendon tissue fibroblasts, bone marrow reticular tissue fibroblasts, other non-epithelial fibroblasts, pericytes, nucleus pulposus cells of the intervertebral disc, odontoblasts/cementoblasts (root-like cementoblast), odontoblast/odontocyte, Hyaline cartilage chondrocytes, fibrocartilage chondrocytes, elastic cartilage chondrocytes, osteoblasts/osteocytes, osteoprogenitors (stem cells of osteoblasts), hyaline cells of the vitreous of the eye, stellate cells of the perilymph space of the ear, hepatic stellate cells (itocytes), pancreatic stellate cells, skeletal muscle cells (e.g., musculoskeletal red muscle cells (slow), musculoskeletal white muscle cells (fast), musculoskeletal intermediate muscle cells, musculoskeletal sack cells of musculus shuttle and nucleus chain cells of musculus shuttle, satellite cells (stem cells), cardiac muscle cells (e.g., normal cardiac muscle cells, sarcomeric cardiac muscle cells and purkinje fibroblasts), smooth muscle cells (various types), muscular epithelial cells of the iris, muscular epithelial cells of the exocrine gland, erythrocytes (erythrocytes), megakaryocytes (platelet precursors), monocytes, connective tissue macrophages(s) of various types, Epidermal langerhans cells, osteoclasts (in the skeleton), dendritic cells (in the lymphoid tissue), microglia cells (in the central nervous system), neutrophils, eosinophils, basophils, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, reticulocytes, stem cells and committed progenitors of the blood and immune system (various types), auditory outer hair cells of the corset organ, basal cells of the olfactory epithelium (stem cells of the olfactory neurons), cold-sensitive primary sensory neurons, heat-sensitive primary sensory neurons, Merkel cells of the epidermis (touch sensor)), olfactory receptor neurons, pain-sensitive primary sensory neurons (different types), photoreceptor cells of the retina in the eye (e.g., photoreceptor rods cells (phototoreceprodcells), Photoreceptor blue-sensitive cone cells of the eye, photoreceptor green-sensitive cone cells of the eye, photoreceptor red-sensitive cone cells of the eye, proprioceptive primary sensory neurons (various types)), touch-sensitive primary sensory neurons (different types), type I carotid body cells (blood PH sensor), type II carotid body cells ((blood PH sensor), type I hair cells of the vestibular apparatus of the ear (acceleration and gravity), type II hair cells of the vestibular apparatus of the ear (acceleration and gravity), type I taste bud cells, cholinergic nerve cells (various types), adrenergic neuron cells (various types, peptidergic nerve cells (various types), inner pillar cells of the organ to be examined, outer pillar cells of the organ to be examined, inner finger cells of the organ to be examined, outer finger cells of the organ to be examined, edge cells of the organ to be examined, Hensen cells of the organ to be examined, Vestibular organ supporting cells, type I taste bud supporting cells, olfactory epithelium supporting cells, Schwann cells (Schwann cells), satellite cells (surrounding the peripheral nerve cell body), enteric glial cells, astrocytes (of various types), neuronal cells (of many types, yet not well classified), oligodendrocytes, spindle neurons, lens pre-epithelial cells, crystallin-containing lens fibroblasts, melanocytes, retinal pigment epithelial cells, oogonic/oocyte, sperm cells, spermatogonic cells (stem cells of spermatocytes), sperm, follicular cells, Sertoli cells (in the testes), thymic epithelial cells and interstitial kidney cells. For example, if the protein of interest expressed in a cell or cell line is an ion channel that is normally expressed in a particular type of neuron, then the reference cell may be that particular type of neuron. Specific types of neurons include, but are not limited to: sensory neurons, neurons of the central nervous system, unipolar neurons, pseudounipolar neurons, bipolar neurons, multipolar neurons, Golgi type I neurons (Golgi I neurons), pyramidal cells, purkinje cells, anterior horn cells, Golgi type II neurons (Golgi II neurons), granulosa cells, basket cells, betz cells, giant motor neurons, medium spiny neurons, ethiocyte, alpha motor neurons, afferent neurons, efferent neurons, motor neurons, and interneurons.

In certain embodiments, the reference cell is a cell of a cell type that normally expresses or functionally expresses the RNA or protein of interest without genetic modification. In certain embodiments, the reference cell is a cell that has the ability to stably express an RNA or protein of interest. In certain embodiments, the reference cell is a cell that has the ability to express an RNA or protein of interest without associated cytotoxicity.

In further aspects, the invention provides methods for producing the cells and cell lines of the invention. In one embodiment, the method comprises the steps of:

a) providing a plurality of cells, at least two of which express an RNA of interest or an mRNA encoding a protein of interest;

c) culturing the cells under a desired set of culture conditions using an automated cell culture method, said culture method characterized in that the conditions are substantially the same for each isolated cell culture, during which culture the number of cells of each isolated cell culture is normalized, and wherein the isolated cultures are passaged according to the same schedule;

d) Determining the isolated cell culture for at least 2 times at least one desired characteristic of the RNA or protein of interest; and

e) identifying an isolated cell culture having the desired characteristics in both assays.

According to this method, the cells are cultured under a desired set of culture conditions. The conditions may be any desired conditions. One of ordinary skill in the art will appreciate which parameters are included within a set of culture conditions. For example, the culture conditions includeWithout limitation: culture medium (basal medium (DMEM, MEM, RPMI, serum-free, serum-containing, fully chemically defined, animal-derived free component), monovalent and divalent ion (sodium, potassium, calcium, magnesium) concentrations, additional components added (amino acids, antibiotics, glutamine, glucose or other carbon sources, HEPES, channel blockers, modulators of other targets, vitamins, trace elements, heavy metals, cofactors, growth factors, anti-apoptotic agents), fresh or conditioned medium, medium with HEPES, pH, specific nutrient depletion or restriction (amino acids, carbon sources)), confluent levels allowed for cells before division/passage, feeder layers for cells, or gamma-irradiated cells, CO ₂Three gas systems (oxygen, nitrogen, carbon dioxide), humidity, temperature, resting or using a shaker, etc., as will be well known to those of ordinary skill in the art.

Cell culture conditions may be selected for convenience or for the particular desired use of the cells. Advantageously, the present invention provides cells and cell lines that are optimally suited for a particular desired use. That is, in embodiments of the invention in which cells are cultured under conditions for a particular desired use, cells are selected that have the desired characteristics under conditions for the desired use.

By way of illustration, if the cells are to be used in a plate in an assay where adhesion of the cells is desired, cells may be selected that show adhesion under the conditions of the assay. Similarly, if the cell is to be used for protein production, the cell may be cultured under conditions suitable for protein production, and selected for advantageous properties for this use.

In certain embodiments, the method comprises the additional step of measuring the growth rate of the isolated cell culture. The growth rate can be determined using any of a variety of techniques well known to those of ordinary skill in the art. Such techniques include, but are not limited to, measuring ATP, cell confluence, light scattering, optical density (e.g., OD260 for DNA). Preferably, the growth rate is determined using a method that minimizes the amount of time the culture spends outside of the selected culture conditions.

In certain embodiments, cell confluence is measured and growth rate is calculated from the confluence value. In certain embodiments, cells are dispersed and clots are removed for improved accuracy prior to measuring cell confluence. Methods for making cells monodisperse are well known and can be achieved, for example, by adding a dispersing agent to the culture to be measured. Dispersants are well known and readily available and include, but are not limited to, enzymatic dispersants such as trypsin, and non-enzymatic cell dissociation agents including, but not limited to, EDTA-based dispersants. Growth rates can be calculated from the confluent date using commercially available software for this purpose, such as HAMILTON VECTOR. Automated confluence measurements, for example using automated microplate readers, are particularly useful. Plate readers for measuring confluence are commercially available and include, but are not limited to, CLONE SELECT IMAGER (Genetix). Typically, at least 2 measurements of cell confluence are made before calculating the growth rate. The number of confluent values used to determine growth rate may be any number convenient or suitable for culture. For example, confluence may be measured multiple times over, for example, 1 week, 2 weeks, 3 weeks, or any time period and at any desired frequency.

When the growth rate is known, according to this method, a plurality of isolated cell cultures are divided into groups by similarity of growth rates. By grouping the cultures into growth rate boxes, the cultures in the groups can be processed together, providing another level of normalization that reduces variation between cultures. For example, cultures in frames may be passaged simultaneously, treated simultaneously with the desired reagents, and so forth. In addition, functional assays generally rely on the cell density in the assay well. In certain embodiments, the true comparison of individual clones is accomplished only by plating them and performing the assay at the same density. Grouping into specific growth rate synchronization groups (cohorts) enables the clones to be plated at a specific density, which allows them to be functionally characterized in a high-throughput format.

The growth rate range in each group may be any convenient range. It is particularly advantageous to select a growth rate range that allows cells to be passaged simultaneously and avoids frequent re-normalization of cell numbers. The growth rate set may include a very narrow range for tight grouping, e.g., average doubling times within 1 hour of each other. But ranges may be as much as 2 hours, as much as 3 hours, as much as 4 hours, as much as 5 hours, or as much as 10 hours, or even broader ranges from each other depending on the method. The need for re-normalization arises when the growth rates in the frames are not the same such that the number of cells in some cultures increases faster than others. To maintain substantially equivalent conditions for all cultures in a box, cells must be periodically removed to re-normalize the number across the box. The less the growth rate, the more frequently renormalization is required.

In step d), cells and cell lines may be tested and selected for any physiological property, including but not limited to: an alteration in a cellular process encoded by the genome; alterations in cellular processes regulated by the genome; a change in a pattern of chromosome activity; a change in a pattern of chromosome silencing; a change in gene silencing pattern; a change in gene activation pattern or efficiency; a change in gene expression pattern or efficiency; alteration of RNA expression pattern or efficiency; changes in RNAi expression pattern or efficiency; alteration of RNA processing pattern or efficiency; alteration in RNA transport pattern or efficiency; a change in protein translation pattern or efficiency; a change in protein folding pattern or efficiency; a change in protein assembly pattern or efficiency; a change in the pattern or efficiency of protein modification; a change in protein transport pattern or efficiency; a change in the mode or efficiency of transport of membrane proteins to the cell surface; a change in growth rate; a change in cell size; a change in cell shape; a change in cell morphology; a change in% RNA content; a change in% protein content; change in% moisture content; changes in% lipid content; a change in ribosome content; a change in mitochondrial content; a change in ER quality; a change in plasma membrane surface area; a change in cell volume; alterations in the lipid composition of the plasma membrane; alteration of lipid composition of the nuclear envelope; changes in the protein composition of the plasma membrane; alteration of the protein composition of the nuclear envelope; a change in the number of secretory vesicles; (ii) a change in the number of lysosomes; a change in the number of cavitation bubbles; alterations in the cells with respect to the following abilities or potentials: protein production, protein secretion, protein folding, protein assembly, protein modification, enzymatic modification of proteins, protein glycosylation, protein phosphorylation, protein dephosphorylation, metabolite biosynthesis, lipid biosynthesis, DNA synthesis, RNA synthesis, protein synthesis, nutrient uptake, cell growth, mitosis, meiosis, cell division, dedifferentiation, conversion into stem cells, conversion into pluripotent cells, conversion into totipotent cells, conversion into stem cell types of any organ (i.e., liver, lung, skin, muscle, pancreas, brain, testis, ovary, blood, immune system, nervous system, bone, cardiovascular system, central nervous system, gastrointestinal tract, stomach, thyroid, tongue, gall bladder, kidney, nose, eye, toenail, hair, taste bud), conversion into any cell type that is differentiated (i.e., muscle, toenail, hair, taste bud), Cardiac muscle, neurons, skin, pancreas, blood, immune, red blood cells, white blood cells, killer T cells, enteroendocrine cells, taste, secretory cells, kidney, epithelial cells, endothelial cells, and also including any of the enumerated animal or human cell types that may be used to introduce nucleic acid sequences), uptake DNA, uptake small molecules, uptake fluorescent probes, uptake RNA, attachment to solid surfaces, adaptation to serum-free conditions, adaptation to serum-free suspension conditions, adaptation to scaled-up cell culture, use in large-scale cell culture, use in drug development, use in high-throughput screening, use in functional cell-based assays, use in membrane potential assays, use in calcium flow assays, use in G protein receptor assays, use in reporter cell-based assays, for use in an ELISA study, for use in an in vitro assay, for use in an in vivo application, for use in a secondary test, for use in a compound test, for use in a binding assay, for use in a panning assay, for use in an antibody panning assay, for use in an imaging assay, for use in a microimaging assay, for use in a multi-well plate, for adaptation to automated cell culture, for adaptation to miniaturized automated cell culture, for adaptation to large scale automated cell culture, for adaptation to cell culture in a multi-well plate (6, 12, 24, 48, 96, 384, 1536 or higher density), for use in a cell chip, for use on a slide, for use on a glass slide, for microarray on a slide or glass slide, for immunofluorescence studies, for use in protein purification, for use in bioproduct production, for use in industrial enzyme production, for use in reagent production for research, for use in vaccine development, for use in cell therapy, for use in transplants or humans, for use in factor isolation secreted by cells, for preparation of cDNA libraries, for purification of RNA, for purification of DNA, for infection by pathogens, viruses or other factors, for resistance to drugs, for suitability maintained under automated miniaturized cell culture bars, for use in protein production for characterization, comprising: protein crystallography, vaccine development, stimulation of the immune system, antibody production, or antibody production or testing. One of ordinary skill in the art will readily recognize suitable tests for any of the above listed characteristics.

Tests that may be used to characterize the cells and cell lines of the invention and/or the matched panel of subjects of the invention include, but are not limited to: amino acid analysis, DNA sequencing, protein sequencing, NMR, testing of protein transport, testing of nuclear mass transport, testing of subcellular localization of proteins, testing of subcellular localization of nucleic acids, microscopy, sub-microscopy, fluorescence microscopy, electron microscopy, confocal microscopy, laser ablation techniques, cell counting, and dialysis. One of ordinary skill in the art understands the method of using any of the above listed tests.

When generating a collection or subject set of cells or cell lines, for example for drug screening, the cells or cell lines in the collection or subject set can be matched such that they are identical (including substantially identical) in one or more selective physiological properties. By "the same physiological property" in this specification is meant that the selected physiological property is sufficiently similar between members of a collection or group of subjects that the collection or group of cells can produce reliable results in a drug screening assay; for example, changes in readings in drug screening assays result from, for example, different biological activities of the test compound on cells expressing different forms of the protein, rather than from changes inherent in the cells. For example, the cells or cell lines may be matched to have the same growth rate, i.e., a growth rate that does not differ by more than 1, 2, 3, 4, or 5 hours between members of a collection of cells or a subject group. In certain embodiments, this can be accomplished by, for example, combining the cell frames into 5, 6, 7, 8, 9, or 10 groups according to their growth rate, and then generating a subject group using cells from the same or different frame-and-group. In certain embodiments, the cell frames may be grouped into 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 groups according to growth rate. In certain embodiments, the cell frames can be grouped into at least 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more than 100 groups depending on the growth rate. In certain embodiments, a subject group of cell lines may include cell lines that are incorporated into the same group based on their growth rate frames. In certain embodiments, a subject group of cell lines may include cell lines that are boxed into different groups based on their growth rate. Methods for measuring cell growth rate are well known in the art. The cells or cell lines in the subject group can also be matched to have the same Z' factor (e.g., a Z factor that differs by no more than 0.1), protein expression levels (e.g., CFTR expression levels that differ by no more than 5%, 10%, 15%, 20%, 25%, or 30%), RNA expression levels, adhesion to tissue culture surfaces, and the like. The matched cells and cell lines can be cultured under the same conditions, for example, obtained by automated parallel processing, to maintain the selected physiological properties. In certain embodiments, the cells or cell lines of the invention can be frame-incorporated into groups based on physiological characteristics of the cells or cell lines, including but not limited to growth rate. In certain embodiments, a matching subject group of cells or cell lines may include cells or cell lines of one or more boxes grouped according to at least one physiological characteristic of the cells, including but not limited to growth rate.

In one embodiment, the subject groups are matched for growth rate under the same set of conditions. Such a panel of subjects (also referred to herein as a matched panel of subjects) is highly desirable for use in a wide range of cell-based studies in which it is desirable to compare the effects of experimental variables between two or more cell lines. Cell lines matched for growth rate maintain substantially the same number of cells per well over time, thereby reducing variations in growth conditions, such as nutrient content between cell lines in the subject group.

According to the present invention, the matched panel may have a growth rate within any desired range, depending on a number of factors, including the characteristics of the cells, the intended use of the panel, the size of the panel, the culture conditions, and the like. Such factors will be readily understood by one of ordinary skill in the art.

The growth rate may be determined by any suitable and convenient method, the only requirement being that the growth rate of all cell lines of a matched panel of subjects be determined by the same method. Numerous methods for determining growth rate are known, as described herein.

The matched panel of subjects of the invention may include any number of clonal cell lines. The maximum number of clonal cell lines in a subject group varies for each use and user and can be as many as can be maintained. In various embodiments, the subject group can include 2, 3, 4, 5, 6, 7, 8, 9, 10, or more clonal cell lines, e.g., at least 12, at least 15, at least 20, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 48, at least 50, at least 75, at least 96, at least 100, at least 200, at least 300, at least 384, at least 400, or more clonal cell lines. In certain embodiments, a matched panel of subjects may include at least 100, 150, 200, 250, 300, 350, 400, 350, 500, 550, 600, 650, 700, 750, 800, 850, 900, or 1,000 clonal cell lines. In other embodiments, the matched panel of subjects may include at least 1,100, 1,250, 1,500, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 clonal cell lines. In other embodiments, the matched panel of subjects may include at least 11,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000 clonal cell lines. In other embodiments, the matched panel of subjects may include at least 100,000, 150,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000 clonal cell lines, or more than 1,000,000 clonal cell lines. In other embodiments, the matched panel may include at least 1,000 clonal cell lines.

According to the present invention, the subject group comprises a plurality of clonal cell lines, i.e., a plurality of cell lines arising from different individual parental cells. In certain embodiments, the plurality of cells in the subject group of cell lines are of the same type. In certain embodiments, the plurality of cell lines in the subject group of cell lines are of at least 2 different types. Any desired cell type can be used to generate a matched panel of subjects. The subject group may include all cell lines of the same cell type or cell lines of different cell types.

The clonal cell lines in the subject panel stably express one or more proteins of interest. Stable expression may be for any period of time suitable for the desired use of the subject group, but at a minimum, long enough to allow selection and use in a matching subject group.

The clonal cell lines of the matched panel may all express the same protein or proteins of interest or some of the clonal cell lines of the panel may express different proteins of interest.

In certain embodiments, the matched panel of subjects includes one or more clonal cell lines expressing different proteins of interest. That is, a first clonal cell line in a subject group can express a first protein of interest, a second clonal cell line in a subject group can express a second protein of interest, a third cell line can express a third protein of interest, etc., corresponding to as many different proteins of interest as desired. The different proteins of interest may be different isoforms of the protein of interest, allelic variants, splice variants or mutations (including but not limited to mutated or truncated sequences), different subunit stoichiometries, different subunit assemblies, differentially folded forms, differentially active forms, forms with different functionalities, forms with different binding properties, forms bound to different cofactors, forms expressed in different cellular contexts, forms expressed in different cellular genetic contexts, forms expressed in cells with different endogenous expression profiles, differentially localized forms, chimeric or chemically modified forms (including enzymatically modified forms, post-translationally modified forms, glycosylated forms, proteolytically modified forms), or combinations thereof. In certain embodiments, the different proteins may be members of a functionally defined group of proteins, such as a bitter taste receptor panel or a kinase panel. In certain embodiments, the different proteins may be part of the same or related signaling pathways. In other subject groups that include heteromultimeric proteins (including heterodimers), the subject group can include two or more different combinations of subunits up to all possible combinations of subunits. Combinations may include subunit sequence variants, subunit isotype combinations, interspecies combinations of subunits, and combinations of subtype forms.

By way of illustration, gamma-aminobutyric acid (GABA) a receptors generally comprise two alpha subunits, two beta subunits and one gamma subunit. There are 6 alpha isoforms, 5 beta isoforms, 4 gamma isoforms and 1 delta, 1 pi and 1 theta and 1 epsilon subunit. The invention comprisesGroups of subjects containing two or more combinations of any of such subunits include groups of subjects containing each possible combination of α, β, γ, δ, π, ε, and θ subunits. In addition, the GABA receptor family also includes GABA_BAnd GABA_CA receptor. The present invention also includes compositions comprising GABA_A、GABA_BAnd GABAc subunits. In certain embodiments, such subject groups comprise human GABA subunits. In other embodiments, the group of mammalian GABA receptor subjects can include a non-human primate (e.g., cynomolgus monkey) GABA receptor, a mouse, rat, or human GABA receptor subject group, or a mixture thereof.

In further examples, the invention relates to one or more epithelial sodium channel (ENaC) subject groups, including any mammalian ENaC subject group such as a non-human primate (e.g., cynomolgus monkey) ENaC, mouse, rat, or human ENaC subject group, or mixtures thereof. Like the GABA receptor, the intact EnaC comprises multiple subunits: α or δ, β and γ. The invention encompasses a panel of subjects having a combination of at least two different ENaC subunits and also includes all possible combinations of ENaC subunits, including combinations of subunits from different species, isoforms, allelic variants, SNPs, chimeric subunits, forms comprising modified and/or unnatural amino acids, and combinations of chemically modified (e.g., enzymatically modified) subunits. The present invention also includes a panel comprising any of the EnaC forms shown in International application PCT/US09/31936, which is incorporated herein by reference in its entirety.

In another embodiment, a matched panel of 25 bitter taste receptors comprises cell lines expressing the natural (unlabeled) functional bitter taste receptors listed in table 10. In certain embodiments, the subject groups are matched for growth rate. In certain embodiments, the subject groups are matched for growth rate and additional physiological properties of interest. In certain embodiments, cell lines in a subject group are generated in parallel and/or screened in parallel.

Experiment ofFurther illustrative but non-limiting examples of object groups and their use are the following: panel of subjects of odorant receptors (insects, dogs, humans, bed bugs), for example for profiling of scents or for discovery of modulators; the panel of cells expressing genes fused to test peptides, i.e. the peptides found to act to internalize cargo (cargo), such as proteins, including monoclonal antibodies or non-protein drugs (cargo may be a reporter molecule such as GFP or AP), into the cells. For this embodiment, the supernatant of cells from this subject group can be added to other cells for evaluation of internalization. In such embodiments, the subject group may include different cell types to assess cell type-specific delivery. Cell line panels expressing different monoclonal antibody heavy chain/light chain combinations were tested to identify active antibodies or monoclonal antibodies. The antibody panel may also provide a series of derivatized forms of monoclonal antibodies to identify those with improved characteristics, such as stability in serum, binding affinity, and the like. Another subject group can be used to express the target protein in the presence of multiple signaling molecules (e.g., different G proteins). Another type of subject panel may be used to test for improved activity/stability of variants of the target protein. The subject panel may include Single Nucleotide Polymorphisms (SNPs) or other mutated forms of the target protein to select for modulators that act on subgroups, many forms, or all forms. Other subject groups can be used to define test compounds for protein families or protein isoforms (e.g., GABA) _AOr other CNS ion channels). The differentially acting compounds can then be used in further studies to determine the function/effect/localization of the corresponding subunit combination in vivo. The test compound may be one that fails a known modulator in the clinic or one that has an adverse off-target effect to determine subunit combinations that may be associated with such effects. Still other subject groups can be used for parallel screening in HTS for reliable assessment of compound activity against various target subtypes to help find compounds active against the desired target and compounds with minimal off-target effects.

The subject panel may include cells that have been engineered to express any desired proteome and all such subject panels are included in the present invention.

The subject group may include cells that have been engineered to express RNA from any desired subject group and all such subject groups are included in the present invention.

In certain embodiments, the invention provides a subject panel of cells or cell lines, wherein the subject panel comprises a plurality of cells or cell lines each expressing a different odorant receptor. Such a panel of odorant receptors can be used to generate an odorant activity profile of a compound or composition of interest. The odor activity profile of a compound or composition refers to the effect of the compound or composition on the activity of multiple odor receptors. To generate an odor activity profile of a compound or composition of interest, the compound or composition is contacted with a plurality of cells or cell lines each expressing a different odor receptor.

In certain embodiments, a subject panel of odorant receptor cells is used to identify compounds that alter, enhance or attenuate an odorant activity profile of a compound or composition. Predicting that a compound identified as altering, enhancing or diminishing an odor activity profile of another compound or composition alters, enhances or diminishes an olfactory effect of the other compound or composition.

In certain embodiments, a panel of odorant receptor cells is used to identify compounds having an odorant activity profile similar to a compound or composition of interest. Compounds identified as having a similar odor activity profile as the compound or composition of interest are predicted to have a similar olfactory effect as other compounds or compositions, i.e., to smell similar or identical. Odor activity spectra can be compared as described below.

Useful odorant receptors include, but are not limited to, the olfactory receptors shown in tables 10-12. In certain embodiments, the odorant receptor is a class I human olfactory receptor and a class II human olfactory receptor, or a combination thereof. The odorant receptor groups of the present invention may have at least 2, 5, 10, 25, 50, 75, 100, 250, 500, 750, 1000, 1500, 2000 or at least 2500 different cells or cell lines each expressing a different odorant receptor. The different odorant receptors may be odorant receptors of different species or of the same species. In certain embodiments, the odorant receptors for use in the cells, groups of subjects, and methods of the invention may be encoded by pseudogenes.

The activity of a compound or composition of interest on an odorant receptor can be measured by any technique known to one of ordinary skill in the art. Assays to measure the effect of a compound or composition of interest on odor receptor activity include, but are not limited to: cell-based assays, fluorescent cell-based assays, imaging assays, calcium current assays, membrane potential assays, high throughput screening assays, fluorescent assays, and combinations thereof. Any assay used in the methods of the invention can be performed in a high throughput format.

Any of the cells or cell lines disclosed herein may be used in the group of subjects that produce the odorant receptor according to the present invention. In certain embodiments, the host cells of the subject panel used to produce the odorant receptor may be cells that have been tested and demonstrated to endogenously express signaling or other protein factors required for functional expression of the odorant receptor. RNA or protein characterization of cells by tests including RT-PCR and microarray methods (including gene chip analysis) can be used to identify such cells.

In certain embodiments, a subject panel of cells of the invention comprises a plurality of cells or cell lines, wherein each cell or cell line has been engineered to express one or more insect odorant receptors. The resulting panel of cells can be used to characterize odorants based on cellular assays, as well as for high throughput screening ("HTS") to identify modulators of odorant receptors.

In certain embodiments, a subject group of cells or cell lines may include all or a subset of odorant receptors from a species (e.g., a mosquito). The subject group of cells or cell lines may comprise at least one odorant receptor from at least two different insect species. Insect scent receptors from any insect species may be used, including insects that transmit human or animal diseases, insects that afflict crops or cause agricultural damage or destruction to plants, including but not limited to mosquitoes, cockroaches, beetles, bed bugs (bed bugs), moths, butterflies, flies, ants, crickets, bees, wasps, fruit flies, ticks (ticks), lice (lice), genetic lice, scorpions, continental horses, centipedes, locusts (grasshoppers), mantises, and spiders. Common methods of identifying proteins as olfactory receptors are based on sequence comparison to known olfactory receptors. In certain embodiments, cell-based assays may also be used to test the reactivity of transmembrane proteins to known volatile or odorous compounds to determine their effect as odorant receptors.

Insect attracting or repelling substances, including compounds and extracts, can be tested against the subject groups provided herein to identify reactive receptors whose activity is modulated by the test compound. The material can be collected from, for example, plants, flowers, food (e.g., cheese), animals, tobacco, waste, secretions, perspiration (including human perspiration, e.g., male perspiration or female perspiration), industrial products, natural and synthetic chemicals, and biological agents. The agent can be tested against a subset or all of the odorant receptor cell lines to identify an activity profile of the compound for all of the test receptors. The activity patterns generated by testing substances against the odorant receptor cell lines can be used for characterization, "fingerprinting" or as diagnostics.

HTS can be used to screen cell lines or panels of subjects containing insect scent receptors for identified responsive substances to identify other substances with similar, enhanced or diminished activity. HTS can be used to screen cell lines or panels of subjects containing insect odorant receptors responsive to substances to identify compounds that modulate, block, or enhance the activity of the receptors in the presence of the substances. HTS can be used to screen cell lines or panels of subjects containing odorant receptors that do not respond to a substance to identify compounds that cause the receptor's response or activity in the presence of the substance.

In certain embodiments, the methods of the invention are used to identify compounds or mixtures thereof that have similar activity against insect odorant receptors as known substances (e.g., known compounds). In more specific embodiments, the methods of the invention are used to identify compounds that mimic the odorant receptor activity of DEET and/or other insect repellents and attractants. In certain embodiments, the methods of the invention are used to identify compounds useful as insect repellents. In certain embodiments, the compounds identified as insect repellents are volatile and non-toxic to the environment, humans, animals, and/or crops. In certain embodiments, the methods of the present invention are used to identify compounds that are useful as insect attractants. In certain embodiments, the compound identified as an insect attractant is a volatile compound and is non-toxic to the environment, humans, animals, and/or crops. Such insect attractants are useful for insect capture. In certain embodiments, the insect attractant or repellent can be specific to a particular insect species.

In particular embodiments, the methods of the invention are used to identify compounds or mixtures of compounds that block the activity of a particular substance on one or more odorant receptors. In more specific embodiments, the methods are used to identify compounds that block a subject's response to sweat, human or animal secretions or components thereof, or carbon dioxide.

In certain embodiments, the methods of the invention are used to identify one or a mixture of compounds that enhance the activity of a particular substance (e.g., a particular compound) on one or more odorant receptors, such as a compound that attracts or repels insects.

In certain embodiments, the methods of the invention are used to identify one or a mixture of compounds that alter the activity of a particular substance (e.g., a particular compound) on one or more odorant receptors. In other embodiments, the methods of the invention can be used to identify a combination of at least 2 compounds, wherein the at least 2 compounds activate the odorant receptor only when combined, and wherein each of the at least 2 compounds does not activate the odorant receptor individually. The receptor may be a receptor for detecting an insect repellent or an insect attractant.

In certain embodiments, the methods of the invention are used to generate odor activity profiles of human sweat samples obtained from males and/or females and fractions of such samples or compounds isolated from such samples to identify reactive receptors, and compare this data with other data (including, for example, results of testing the same substance for one or more insect species) to identify activities or compounds associated with insect repellence or attraction.

In certain embodiments, the methods of the present invention are used to test and compare odor activity profiles of at least two samples. In particular embodiments, the samples may be volatiles obtained from different plants, different species of plants or animals, or from different flowers. The odor activity profiles of the different samples are then compared to identify the response receptors (e.g., those whose activity is affected by the different samples). The reactive receptor is an indicator of the chemical composition of the sample. The similarity of the odor activity profiles of the samples to be compared is a measure of the chemical similarity of the different samples.

In certain embodiments, the methods of the invention are used to identify a number of chemically distinct compounds with similar activities, which are used in combination or sequentially or introduced to address potential insect adaptations or evolutions that may render at least one compound ineffective.

A panel of subjects each comprising one or more human odorant receptor cell lines can be generated for cell-based assays to characterize odorants and for HTS to identify odorant receptor modulators.

A substance comprising a compound or extract having an aroma, odor, fragrance, or aroma can be tested against a panel of human odorant receptors to identify reactive receptors whose activity is modulated by the test substance. Compounds or mixtures can be collected from plants, flowers, food, animals, cigarettes, tobacco, truffles, musk (musk), herbs, mints, waste, secretions, perspiration (including human perspiration, such as male or female perspiration), industrial products, natural and synthetic chemicals, and biologicals (including diseased tissues and tumors). Compounds can be tested against a subset or all of the human odorant receptor cell lines to identify an activity profile of the compound for all of the tested receptors. The activity patterns generated by testing substances against human odorant receptor cell lines can be used for characterization, "fingerprinting" or as diagnostics.

HTS can be used to screen cell lines or panels of subjects that include human odorant receptors identified as responsive substances to identify other substances with similar, enhanced or diminished activity. HTS can be used to screen cell lines that include human odorant receptors responsive to substances to identify compounds that modulate, block, or enhance the activity of the receptors in the presence of the substances. HTS can be used to screen cell lines that include human odorant receptors that do not respond to a substance to identify compounds that cause a receptor response or activity in the presence of the substance.

Exemplary uses of the odor receptor panel include:

identifying one or a mixture of compounds having activity towards one or more odour receptors similar to known substances, for example synthetic compounds which mimic the activity of musk.

Identifying one or a mixture of compounds that block the activity of a known substance on one or more odour receptors, for example a compound that blocks a substance with malodour (human or animal perspiration).

Identifying one or a mixture of compounds that enhance the activity of a known substance on one or more odorant receptors, such as a compound that mimics the aroma of roses or the aroma of beefsteaks.

Identifying one or a mixture of compounds that alter the activity of a known substance on one or more odorant receptors, for example a compound that leads to the activation of a receptor for detecting malodour in the presence of a substance (e.g. tobacco smoke) (which may not normally produce this effect).

One or a mixture of compounds that alter the activity of a known substance on one or more odorant receptors, for example a compound that results in the activation of a receptor that detects a desired aroma in the presence of a substance (e.g., a malodorous substance), such as a substance that can typically activate other receptors without activating the receptor corresponding to the desired aroma, is identified.

Human sweat samples obtained from men and women and fractions of such samples or compounds isolated from such samples are characterized to identify reactive receptors, and this data is correlated with other data (including, for example, human psychophysical studies) to identify activities or compounds that correlate with perceived malodor or attraction.

A set of samples that can be compared (e.g., aromas from different types of roses, or aromas from different flowers or fractions of a substance) are tested to identify common or specific receptors that respond to such aromas.

The cells, subject groups, and methods described herein can be used for odorant receptors from other species, including but not limited to: a mammalian species selected from the group consisting of: humans, non-human primates, bovines, porcines, felines, rats, marsupials, murines, canines, ovines, caprines, rabbits, guinea pigs, and hamsters; or an insect species selected from the group consisting of: the family of the Mosquidae, including the genera Anopheles (Onychidae and all mosquito subspecies), Aedes (including Aedes aegypti and all subspecies), Culex and Aedes, gnat, Black fly, Chrysomyiame, Siphona, Philippinensis, Siphonia, Hemiptera, Dermaptera, Primulus, Dermaptera, and Digitalis (lygus), Anserida, Siphonia, Pediculus, Humanis, lice, Ornithoechia, Eye Gnatures, stable, deer and horse fly, aphid, plant hopper, snout moth, borer, Lepidoptera, nematoda, Homoptera, Coleoptera, Termite (Mites), Termite, Blastoma, Blatta cockroaches (Germany cockroaches), Cypridae, Raynaud, Haemaphyceae, Haemaphysalidae, Phryptotheca, Phryptophyceae, Phryptolitha, Philippinaceae, Philippinard, Philidae, Philippines, Philippinaceae, Orthoides, Orthodes, Ornithocidomyia, Ornithokes, Ornithocidae, Ornithodes, Whitefly, diabrotica, tenebrionidae, weevil superfamily, cereal weevils (guinweill), cereal weevils (Sitophilus granaria), rice weevils (rice weevil), rice weevils (Sitophilus oryzae), alfalfa leaf weevils, fruit core weevils on peas, pea weevils on bean seeds, phaseolus vulgaris, tetranychidae, rhinotermitaceae, kolotermitadae, cockroaches (blatellideae), formidae, beetles and bugs, and e.

The odor activity spectrum of the compound of interest may be compared to the signature odor activity spectrum by calculating a relationship between odor activity spectra, such as, but not limited to, calculating a similarity measure between odor activity spectra. The signature odor activity profile may be one of a set of odor activity profiles in a database. The characteristic odor activity profile may be a historical profile. The odour activity profile of a compound of interest may comprise measurements representing the effect of the compound on the activity of two or more odour receptors in a group of subjects. Each signature odorant activity spectrum includes measurements representing the effect of each compound on the activity of two or more odorant receptors in a group of subjects.

In certain embodiments, individual compounds corresponding to a characteristic odor activity spectrum may be associated with known odors. That is, the measurements of the effect of each compound on the activity of two or more odorant receptors in a subject group can be combined to generate a signature transcript profile (transcript profile) that can be correlated with known odorants. A database of the signature odor activity spectrum may be stored on a computer readable storage medium. In particular embodiments, the database comprises at least 10 characteristic odor activity spectra, at least 50 characteristic odor activity spectra, at least 100 characteristic odor activity spectra, at least 500 characteristic odor activity spectra, at least 1,000 characteristic odor activity spectra, at least 10,000 characteristic odor activity spectra s, or at least 50,000 characteristic odor activity spectra, each characteristic odor activity spectrum comprising at least 2, at least 10, at least 100, at least 200, at least 500, at least 1,000, at least 2000, at least 2500, at least 7500, at least 10,000, at least 20,000, at least 25,000, or at least 35,000 component measurements. In the above embodiments, the odor activity spectrum of the compound of interest can be compared to a plurality of signature odor activity spectra to determine one or more signature odor activity spectra that correlate with (e.g., are most similar to) the odor activity spectrum of the compound of interest, and the compound of interest can be characterized as having a known odor associated with each compound corresponding to such one or more signature odor activity spectra.

In other embodiments, the compound of interest may be associated with a known odor. In the above embodiments, the odor activity spectrum of the compound of interest can be compared to a plurality of signature odor activity spectra to determine one or more signature odor activity spectra that are correlated to (e.g., most similar to) the odor activity spectrum of the compound of interest, and each compound corresponding to such one or more signature odor activity spectra can be characterized as having a known odor that is correlated to the compound of interest.

In certain embodiments, the relationship between the odor activity profile of the compound of interest and each of the plurality of hallmark odor activity profiles stored in the database can be calculated. The correlation may be calculated by comparing a measure in the odour activity spectrum of the compound of interest representing the effect of the compound of interest on the selected activity of two or more odour receptors in the subject group with a measure in the corresponding signature odour activity spectrum representing the effect of different compounds on the same selected activity of two or more odour receptors in the subject group. An odor activity spectrum of a compound of interest can be considered to be associated with a characteristic odor activity spectrum if the characteristic odor activity spectrum is measured to be within about 2%, about 5%, about 8%, about 10%, about 12%, about 15%, about 20%, about 25%, about 30%, or about 35% of the odor activity spectrum of the compound of interest.

The odor activity spectrum of the compound of interest is considered to be most similar to the hallmark odor activity spectrum if the measure of similarity between the odor activity spectrum and the hallmark odor activity spectrum of the compound of interest is above a predetermined threshold. In particular embodiments, the predetermined threshold may be determined as a value representing a measure of similarity in the hallmark odor activity spectrum measured within about 2%, about 5%, about 8%, about 10%, about 12%, about 15%, about 20%, about 25%, about 30%, or about 35% of the odor activity spectrum of the compound of interest.

In certain embodiments, the odor activity spectrum of a compound of interest can be expressed as a vector p,

p＝[p₁，...p_i，...p_n]

wherein p is_iIs a measure of the ith component, e.g., the effect of the compound of interest on the ith biological activity of a given odorant receptor in a subject group. In specific embodiments, n is greater than 2, greater than 10, greater than 100, greater than 200, greater than 500, greater than 1000, greater than 2000, greater than 2500, greater than 7500, greater than 10,000, greater than 20,000, greater than 25,000, or greater than 35,000. Each signature odor activity spectrum can also be represented as a vector p. In calculating the correlation, for each component i 1.. n, the measured quantity of the ith component in the vector representing the odor activity spectrum of the compound of interest can be compared with the measured quantity of the ith component of the corresponding vector representing the characteristic odor activity spectrum.

The correlation can be calculated according to the method of the invention using any statistical method known in the art for determining the probability of correlation of two data sets to identify whether a correlation exists between the odor activity profile of the compound of interest and the signature odor activity profile. For example, the correlation between the odor activity spectrum (pi1) of the compound of interest and each hallmark odor activity spectrum (pi2) can be calculated using the similarity measure sim (pi1, pi 2). One way to compute the similarity measure sim (pi1, pi2) is to compute the euclidean distance to the negative 2 th power. In alternative embodiments, sim (pi1, pi2) may be calculated using a metric other than euclidean distance, such as manhattan distance, chebyshev distance, vector angle, correlation distance (correlation distance), normalized euclidean distance (normalized euclidean distance), mahalanobis distance, squared pearson correlation coefficient, or minkowski distance. In certain embodiments, a pearson correlation coefficient, squared Euclidean distance, sum of squared Euclidean (Euclidean sum of square), or squared pearson correlation coefficient is used to determine similarity. Such measures can be calculated, for example, using AS (statistical analysis Systems Institute, Cary, North Carolina) or S-Plus (statistical sciences, Inc., Seattle, Washington). The use of such measures is described in Draghici, 2003, Data Analysis Tools for DNA microarray, Chapman & Hall, CRC Press London, Chapter 11, which is hereby incorporated by reference in its entirety for this purpose.

The correlation may also be calculated based on rank, where x_iAnd y_iIs the ranking of the measured values in ascending or descending numerical order. See, e.g., Conover, practical Nonparametric statics, 2 nd edition, Wiley, (1971). Shannon mutual information may also be used as a similarity measure. See, for example, Pierce, An Introduction To information theory: symbols, Signals, and Noise, Dover (1980), incorporated herein by reference in its entirety.

Various classifiers known in the art can be trained according to the methods described in this application and used to classify the compound of interest as having an odor. The algorithm can be used to generate a classifier that can predict the odor of a compound of interest using the odor activity spectrum of the compound of interest.

In certain embodiments, the trainable classifier classifies a compound according to odor using a measure in the signature odor activity spectrum of a previously characterized compound and a known odor associated with the previously characterized compound. Individual compounds corresponding to a characteristic odor activity spectrum may be associated with known odors. The classifier may be an algorithm for classification by using an unsupervised or supervised learning algorithm for estimating the measured and known odors in the signature odor activity spectrum of previously characterized compounds. The odor activity spectrum of a compound of interest can be processed using a classifier to classify the compound of interest according to odor. That is, a classifier can be used to classify a compound of interest as having one or more known scents associated with a plurality of hallmark scent activity profiles used to train the classification.

In certain embodiments, the compound of interest can be associated with a known odor. In the above embodiments, the classifier may be trained to identify one or more signature odor activity profiles that may be correlated with a known odor of a compound of interest based on the odor activity profile of the compound of interest. The classifier may be an algorithm for classification by using an unsupervised or supervised learning algorithm for estimating the measurands in the signature odor activity spectrum of each compound and identifying one or more signature odor activity spectra that correlate to a known odor of the compound of interest based on the odor activity spectrum of the compound of interest.

Any standard unsupervised or supervised learning technique known in the art may be used to generate the classifier. The following are non-limiting examples of unsupervised and supervised algorithms known in the art. In view of the disclosure of the present application, one of ordinary skill in the art will appreciate that other pattern classification or regression techniques and algorithms may be used for the classifier and the present invention includes all such techniques.

A neural network. In certain embodiments, the classifier learns using a neural network. Neural networks are two-stage regression (two-stage regression) or classification decision rule (classification decision rule). The neural network has a layered structure including a layer of input cells (and bias cells) connected to a layer of output cells through a layer of loads. With respect to regression, the output cell layer typically includes only one output cell. However, neural networks can handle multiple quantitative reactions in a seamless manner.

In a multilayer neural network, there are an input cell (input layer), a hidden cell (hidden layer), and an output cell (output layer). Further, there is a single deviation unit connected to each unit except the input unit. Neural networks are described in duca et al, 2001, pattern classification, second edition, John Wiley & Sons, inc., New York; and Hastie et al, 2001, The Elements of Statistical Learning, Springer-Verlag, New York, each of which is incorporated by reference herein in its entirety. Neural networks are also described in Draghici, 2003, Data Analysis Tools for DNA microarray, Chapman & Hall/CRC; and Mount, 2001, Bioinformatics: sequence and genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, each of which is incorporated herein by reference in its entirety. Discussed below are some exemplary forms of neural networks.

The basic approach to using neural networks is to start with an untrained network, present a training pattern to the input layer, then pass the signal through the network and measure the output at the output layer. These outputs are then compared to target values; any difference corresponds to an error. For classification, the error may be a squared error or a cross entropy (bias). See, for example, Hastie et al, 2001, The Elements of Statistical Learning, Springer-Verlag, New York, which is incorporated by reference herein in its entirety.

The 3 commonly used training schemes are random, batch and on-line training. In random training, patterns are randomly selected from a training set and the network weights presented by each pattern are updated. A multi-layer nonlinear network trained by a gradient descent method, such as a random back-propagation method, performs maximum likelihood estimation of weights in a classifier determined by the network topology. In batch training, all patterns are presented to the network before learning occurs. Typically, in batch training, several approaches are generated through training data. In online training, models are presented to the network once and only once.

A problem that often arises in three-tier network usage is the optimal number of concealed elements for the network. The number of inputs and outputs of a three-layer network is determined by the problem to be solved. In the present invention, the number of inputs to a given neural network will equal the number of biomarkers selected from Y. The number of outputs of the neural network will typically be exactly 1. If too many hidden units are used in the neural network, the network will have too many degrees of freedom and if the training time is too long, there will be a risk that the network will over-fit the data. If there are too few concealed units, the training set cannot be learned. However, it is generally better to have too many concealed units than to have too few concealed units. With too few hidden units, the classifier may not have sufficient flexibility to capture non-linearities in the data; with too many concealed units, the additional weight can be reduced to 0 if appropriate regularization or pruning as described below is used. In a typical implementation, the number of concealment units is some number in the range of 5 to 100, the number increasing as the number of inputs and the number of training cases increases.

And (6) clustering. In certain embodiments, the classifier uses clustering for learning. In certain embodiments, selection component i of the vector representing the signature odor activity profile is used to cluster the odor activity profiles. In certain embodiments, prior to clustering, the measurements are normalized to have a mean and unit variance of 0.

The signature odor activity profiles that showed similar measured patterns throughout the training population tended to cluster together. When the vectors form clusters according to a particular known smell, the particular combination of components i that are measured can be considered a good classifier in this aspect of the invention. See, for example, page 211-. As described in section 6.7 of duca 1973, the clustering problem is described as one of the natural groupings found in the data set. To identify natural groupings, 2 problems are to be solved. First, a method of measuring the similarity (or dissimilarity) between two odor activity profiles is determined. This measure (similarity measure) is used to ensure that the odor activity profiles in one cluster are more similar to each other than they are to other odor activity profiles. Second, a mechanism for assigning data into clusters using a similarity measure is determined.

Similarity measures are discussed in section 6.7 of Duda1973, where one method proposed to begin clustering studies is to determine a distance function and calculate a matrix of distances between pairs of odor activity spectra. If the distance is a good measure of similarity, the distance between the odor activity spectra in the same cluster is significantly smaller than the distance between the odor activity spectra in different clusters. However, as mentioned in Duda1973, page 215, clustering does not require the use of a distance metric. For example, a non-metric similarity function s (x, x ') may be used to compare two vectors x and x'. Conventionally, s (x, x ') is a symmetric function whose value is maximum when x and x' are "similar" in some way. An example of a non-metric similarity function s (x, x') is provided on page 216 of duca 1973.

Once the method of measuring "similarity" or "dissimilarity" before a point of the data set has been selected, clustering requires a standard function of measuring the clustering quality of any partition of data. Partitions of the standard functionally-polarized dataset are used to cluster the data. See, for example, Duda1973, page 217. The criteria function is described in section 6.8 of duca 1973. More recently, Duda et al, Pattern Classification, 2 nd edition, John Wiley & Sons, Inc. New York has been published. Page 537-563 describes the clustering in detail. Additional information on clustering techniques can be found in Kaufman and Rousseeuw, 1990, filing Groups in Data: introduction to Cluster Analysis, Wiley, New York, NY; everett, 1993, Cluster analysis (3 rd edition), Wiley, New York, NY; and Backer, 1995, Computer-Assisted reading in Cluster Analysis, Prentice Hall, UpperSaddle River, N.J.. Specific exemplary clustering techniques that may be used with the present invention include, but are not limited to, hierarchical clustering (agglomerative clustering using nearest neighbor, farthest neighbor, average linkage, centroid, or sum-of-squares algorithms), K-means clustering, fuzzy K-means clustering, and Jarvis-Patrick clustering.

And (4) analyzing a main component. In certain embodiments, the classifier is learned using principal component analysis. Principal component analysis is a classical technique that reduces the dimensionality of a data set by transforming the data into a set of new variables (principal components) that summarize the features of the data. See, for example, Jolliffe, 1986, Principal Component Analysis, Springer, New York, which is incorporated by reference herein in its entirety. Principal component analysis is also described in Draghici, 2003, Data analysis tools for DNA microarray, Chapman & Hall/CRC, which is incorporated herein by reference in its entirety. The following are non-limiting examples of principal component analysis.

The Principal Components (PCs) are uncorrelated and ordered such that the kth PC has the kth largest variance between PCs. The kth PC may be interpreted as the direction that maximizes the variance of the projection of the data point so that it is orthogonal to the kth-1 PC. The first few PCs captured most of the variance in the dataset. Instead, it is generally assumed that the last few PCs only capture the residual 'noise' in the data.

In one approach using a PCA learning classifier, vectors representing signature odor activity spectra can be constructed in the same manner as described above for clustering. In fact, the set of vectors where each vector represents a characteristic odor activity spectrum may be considered as a matrix. In certain embodiments, the matrix represents a qualitative binary description of the monomers in the frierson method (Kubinyi, 1990, 3D QSAR in drug design methods and applications, Pergamon Press, Oxford, pp 589-.

Each vector, where each vector represents a member of the training population (e.g., a signature odor activity profile), is then plotted. Many different types of graphs are possible. In certain embodiments, a one-dimensional map is made. In the one-dimensional plot, the values of the first major component from each member of the training population are plotted, with the expectation that the odor activity spectra corresponding to the odors will cluster within one range of the first major component values and the spectra corresponding to another odor will cluster within a second range of the first major component values.

In certain embodiments, members of the training population are mapped against more than one principal component. For example, in certain embodiments, the members of the training population are plotted on a two-dimensional graph, where the first dimension is the first principal component and the second dimension is the second principal component.

Nearest neighbor analysis. In certain embodiments, the classifier is learned using nearest neighbor analysis. The nearest neighbor classifier is memory based and does not require a classifier fit. Given a query point x₀Identification of distance from x₀The nearest k training points x_(l)R.. k, then classify point x using k nearest neighbor₀. The contact may be interrupted randomly. In certain embodiments, the euclidean distance in feature space is used to determine the distance as:

d_(i)＝||x_(i)-x_o||。

Generally, when using the nearest neighbor algorithm, the abundance data from Y used to calculate the linear discrimination is normalized to have a mean of 0 and a variance of 1. In the present invention, members of the training population are randomly assigned to the training and test groups. For example, in one embodiment, members of the training population of 2/3 are placed into the training group and members of the training population of 1/3 are placed into the test group. The selected combination of vector components i represents the feature space in which the members of the training set are plotted. The ability of the training set to correctly characterize the members of the test set is then calculated. In some embodiments, the nearest neighbor calculation is performed several times for a given combination of vector components i. In each iteration, members of the training population are randomly assigned to the training and test groups.

The nearest neighbor rule may be refined to handle unequal class priors, unequal class misclassification costs, and feature selection. Many such improvements include some form of weighted voting for neighbors. For more information on nearest neighbor analysis, see, e.g., duca, Pattern Classification, 2 nd edition, 2001, John Wiley & Sons, Inc; and Hastie, 2001, The Elements of Statistical Learning, Springer, New York, which is hereby incorporated by reference in its entirety.

And (5) linear discriminant analysis. In certain embodiments, linear difference analysis is used to learn the classifier. Linear Discriminant Analysis (LDA) attempts to classify subjects into one of two categories based on certain target properties. In other words, the LDA tests whether the target property measured in the experiment predicts the classification of the target. LDA usually requires continuous arguments and discontinuous class dependent variables (variants). In the present invention, abundance values of selected combinations of vector components i in a subset of the entire training population are used as the necessary continuous independent variables. The trait subgroup classification (e.g., odor) of each member of the training population is used as a discrete class dependent variable.

LDA finds a linear combination of variables that maximizes the ratio of inter-group variance to intra-group variance by using grouping information. Clearly, the linear weights used by LDA depend on the degree to which the vector component i is measured dispersed in the odor group throughout the training set. In certain embodiments, LDA is used to train a data matrix of members in a population. The linear discriminants for each member of the training population are then plotted. Ideally, those members of the training population that represent a scent will cluster to one range of linear discriminant values (e.g., negative) and those members of the training population that represent another scent will cluster to a second range of linear discriminant values (e.g., positive). LDA is considered more successful when the separation between clusters of discrimination values is greater. For more information on linear discriminant analysis, see, e.g., duca, Pattern Classification, 2 nd edition, 2001, John Wiley & Sons, Inc; and Hastie, 2001, The Elementsof Statistical Learning, Springer, New York; and Venables & Ripley, 1997, Modern Applied Statistics with s-plus, Springer, New York, each of which is incorporated herein by reference in its entirety.

And (5) performing secondary discriminant analysis. In certain embodiments, the classifier is learned using quadratic discriminant analysis. Quadratic Discriminant Analysis (QDA) takes the same input parameters and returns the same results as LDA. QDA uses quadratic equations rather than linear equations to produce results. LDA and QDA are interchangeable, the choice and/or availability of which software to use to support the analysis is a matter of choice. Logistic regression takes the same input parameters and returns the same results as LDA and QDA.

And a support vector machine. In some embodiments, a support vector machine is used to learn the classifier. SVMs are described, for example, in crisiatini and Shawe-Taylor, 2000, An Introduction to support Vector Machines, Cambridge University Press, Cambridge; boser et al, 1992, "A training algorithm for optimal markers", Proceedings of the 5^thAnnual ACM Workshop on computerized learning Theory, ACM Press, Pittsburgh, PA, pp.142-152; vapnik, 1998, Statistical Learning Theory, Wiley, New York; mount, 2001, Bioinformatics: sequence and genome analysis, Cold Spring Harbor laboratory Press, Cold Spring Harbor, New York, Duda, Pattern Classification, 2 nd edition, 2001, John Wiley &Sons, inc; and Hastie, 2001, The Elements of Statistical Learning, Springer, New York; and Furey et al, 2000, Bioinformatics 16, 906-914, each of which is incorporated by reference herein in its entirety. When used for classification, the SVM separates a given set of binary label data training data from the hyperplane that is the farthest from them. For cases where non-linear separation is not possible, SVMs may be used to automatically implement non-linear mapping to the feature space. The hyperplane found in the feature space by the SVM corresponds to a non-linear decision boundary in the input space. AboutFor more information on support vector machines, see, e.g., Furey et al, 2000, Bioinformatics 16, pages 906 and 914, which are incorporated herein by reference in their entirety.

And (4) a decision tree. In one embodiment, the classifier is a decision tree. Decision trees are generally described in Duda, 2001, Pattern Classification, John Wiley & Sons, Inc., New York, pp.395-396, which is incorporated herein by reference in its entirety. One specific algorithm that can be used is classification and regression trees (CART). Other specific algorithms include, but are not limited to, ID3, C4.5, MART, and Random forest (Random forest). CART, ID3 and C4.5 are each described in Duda, 2001, Pattern Classification, John Wiley & Sons, Inc., New York, pp. 396-408 and 411-412, which are incorporated herein by reference in their entirety. CART, MART and 4.5 are also described in Hastie et al, 2001, The Elementsof Statistical Learning, Springer-Verlag, New York, Chapter 9, which is incorporated herein by reference in its entirety. The Random forms technique is described in Breiman, 1999, "Random forms-Random Features", Technical Report 567, StatisticDepartment, University of California at Berkeley, September 1999, which is incorporated herein by reference in its entirety.

The classifier may be a multivariate decision tree, except for a univariate decision tree in which each split (split) is based on the measurands of the corresponding vector component i or the relative measurands of the vector component i. In such a multivariate decision tree, some or all of the decisions actually comprise a measured linear combination of a plurality of vector components i. Multivariate decision trees are described in Duda, 2001, Pattern Classification, John Wiley & Sons, Inc., New York, pp.408-409, which is incorporated herein by reference in its entirety.

Multivariate adaptive regression splines (Multivariate adaptive regression splines). Another method that may be used to learn the pairwise probability function gpq (X, Wpq) uses the multivariate adaptive regression spline Method (MARS). MARS is an adaptive process for regression and is well suited to the high-dimensional problem posed by the present invention. MARS can be viewed as a generalization of stepwise linear regression or an improvement of the CART method to improve the performance of CART in a regression setting. MARS is described in Hastie et al, 2001, The Elements of Statistical Learning, Springer-Verlag, New York, pp.283-295, which is incorporated herein by reference in its entirety.

Gravity classifier techniques. In one embodiment, a nearest neighbor centroid classifier technique is used. For different odors, such a technique calculates the center of gravity given by the average of the vector components i in the training population (signature odor activity spectrum) measured, and then assigns the vector representing the compound of interest to the class whose center of gravity is closest. This method is similar to k-means clustering except that clusters are replaced by known classes. An exemplary implementation of this method is predictive analysis or PAM of microarrays. See, e.g., Tibshirani et al, 2002, Proceedings of the National Academy of science USA 99; 6567 and 6572, which are incorporated herein by reference in their entirety.

And (6) regression. In certain embodiments, the classifier is a regression classifier, such as a logistic regression classifier. Such a regression classifier includes coefficients for constructing each odor activity spectrum of the classifier. In such embodiments, the coefficients of the regression classifier are calculated using, for example, a maximum likelihood method. In such calculations, the measured amount of carrier component i is used.

Other methods. In certain embodiments, the classifier is learned using k-nearest neighbor (k-NN), Artificial Neural Networks (ANNs), parametric linear equations, parametric quadratic equations, naive Bayes analysis (naiveBayes analysis), linear discriminant analysis, decision trees, or Radial basis functions (Radial basis functions).

Some embodiments of the invention provide computer program products including any or all of the program modules shown in fig. 1. Aspects of the program modules are described further below.

In certain embodiments, the invention provides a cell or cell line, or a panel of cells or cell lines, that expresses a biological product, such as a secreted protein. Secreted proteins may include antibodies or active fragments thereof, e.g., proteins comprising heavy and light chains, single chain antibodies, proteins active in the immune system, IgA, IgD, IgE, IgG and IgM proteins or active fragments thereof, enzymes, coagulation factors or hormones or fragments of any such corresponding proteins. Biologicals may also include FDA approved biologicals drugs, known biologicals, proteins with therapeutic activity, or therapeutic biologicals. Examples of biologicals and their trade names include, but are not limited to: carnanitumumab (Canaki) (Maris), botulinum toxin A (Dysport), Golimumab (Golomumab) (Simponi), romidept (Romiprostim) (NPLATE), Cetoluzumab (Certoluzumab Pegol) (Cimzia), Leluocipu (Rilonacept) (Arcalalyst), methoxypolyethylene glycol-betalaiptin (Mirrea), Ekulizumab (Soliris), panitumumab (Vectibix), Idubulase (Elaprase), Ranibizumab (ranis), Arozolosidase alpha (Myozyme), Abasipul (Orencia), Gansula (Naglazyme), Rivelumab (Kelvizumab), Kelvin (Kelvibrinoglobin), Ab (Tyr-Ab), Ab (Avalvimentin), Ab (Ab-Ab), Ab (Ab-Ekulizumab), Ab (Versatic-a), Ab (Ab-a) (Versatic), Ab (Ab-Ab) (Versatic) (Ab) (Versatic) Ab) (Ab) Ab-Ab (Ekuromavil) (Ab) and Ab (Ab) Ab (Ab), Alfa bepotastine ((Aranesp), urokinase (yakinase), ibritumomab tiuxetan (zerumab), rituximab (rituximab), aldesleukin (Proleukin), dinierein (Ontak), pemetrexed (pegapase), filgrastim (Neupoen), ompreinterleukin (neumagea), pefilgrasta (Nuelasta), sargrastim (Leukine), palivumin (Kepivance), trastuzumab (Herceptin), cetuximab (eribix), asparaginase (Elspar), labirise (Elitek), alemtuzumab (Campath), tositumomab (Bexxar), palivizumab (Synagis), interferon alpha-2 a (Roferon-a), interferon alpha-2 b (pegg-lntopas), interferon alpha-2 a (pegaptane-2 a), interferon alpha-2 a (peginterferon alpha-2 a), interferon alpha-2 a (peggepeggepegaptane), interferon alpha-2 a (peggepeggephyra), interferon alpha-2 a (pegapten-2 a), interferon alpha-2 a, pegapten alpha-interferon alpha-2 a (pegapten-2 a), pegapten alpha-b, pegapten alpha-interferon alpha-2 a, pegapten, pegapta, pegapten alpha-, Daclizumab (Zenapax), basilix (Simulect), Moromona-CD 3(Orthocolone, OKT3), interferon- γ 1B (Actimmune), tegaserod alpha-activated (Xigris), collagenase (Santyl), Becapeline (Regranex), Efalizumab (Raptiva), Alleaux (Amevive), interferon alpha-N3 (Alferon N), sulfatase (Naglazyme), acarbose beta (Fabrazyme), Raraniase (Aldurazyme), Enlix (Remicade), Abarex (Orencia), Anacardia hysteresis (Kineret), adalimumab (Humira), Ensizumab (Enbrel), Xylarius (Xylair), nucleic acid (Tynilla), botulinum alpha-interferon alpha (botulinum alpha), botulinum beta-interferon alpha-activated (B-1-B), botulinum toxin beta (botulinum beta-1B), botulinum beta-interferon beta (botulinum beta-B), botulinum toxin (botulinum beta-type B-interferon beta-1B), botulinum beta-interferon beta-N (interferon beta-B), and (botulinum toxin B-B), or (botulinum toxin B, gamma-B, gamma-interferon alpha-beta-interferon (interferon, gamma, Interferon beta-1-a (avonex), tenecteplase (TNKase), streptokinase (Streptase), reeplase (retavase), abciximab (ReoPro), tenecteplase (Cathflo activise, activin), epo alpha (abeamed, Binocrit), idose-2-sulfatase (elapraprase), insulin (Exubera), HPV vaccine (Gardasil), pegaptanib (pegaptanib) (Macugen), human acid-a-glucosidase (Myozyme), thiolase (Naglazyme), human growth hormone (omnicope), parathyroid hormone (preotech), human growth hormone (Valtropin), antithrombin (Atryn), major capsid L1 protein (Cervarix), erythropoietin alpha (hexoxime), Epsiloxamine (Epstein), human methoxyphage-1-IGF (macrogol), macrogol (macrogol/macrogol), macrogol (interferon alpha/macrogol), macrogol (interferon beta-1-IGF (macrogol), macrogol (macrogol/macrogol), macrogol (interferon alpha/macrogol), macrogol (interferon alpha, macrogol), macrogol (macrogol), macrogol (macro, Silapo), ribavirin and interferon alpha-2 b (Rebetron combination therapy), arabinocerebrosidase (Cilizyme), imidurinase (Cerezyme), human insulin (Youngin, Nutropin, nordherin), somatotropin (Humatrope, Nutropin/Nutropin AQ), semorelin (Geref), human methionine auxin (Protopin), human albumin (Albutein), and gemtuzumab ozogale (Mylotarg).

Cells and cell lines having properties most suitable for expression of secreted proteins can be selected using known tests that characterize clones in terms of any of a variety of properties, including but not limited to: post-translational processing or modification, yield, percent active product, stability of certain properties (e.g., by testing the properties described herein over time), and stoichiometry (e.g., by RT-PCR or protein analysis).

Post-translational processing or modification of a secreted protein can be characterized by assays known in the art, including but not limited to protein sequencing, mass spectrometry, methods of testing for glycosylation or phosphorylation, or covalent addition of chemical groups or residues, to select cell lines and conditions that result in a particular or desired form of the secreted protein product. Cells and cell lines can be designed or selected to express one or more factors that affect post-translational processing or modification of a secreted product, for example by introducing sequences corresponding to enzymes that catalyze post-translational processing or modification or by testing cell lines to select cell lines that endogenously express such enzymes or are genetically activated to express such enzymes.

Cells and cell lines with the greatest production or yield of secreted product can be produced by testing isolated clones using methods that estimate the level of secreted protein product, for example, using ELISA methods (e.g., ELISA to detect FC fragments to estimate antibody yield).

Cells and cell lines that result in the greatest percentage of active secreted protein compared to the total production of secreted product can be produced by using methods that estimate the activity or binding of the protein, e.g., by testing isolated clones using activity assays, functional assays or binding assays, e.g., functional cell-based assays, ELISA using capture reagents comprising binding epitopes, secondary assays, animal studies, or assays for measuring binding or activity of the protein.

Cell lines additionally optimized for growth in animal component-free media conditions and/or suspension conditions (as used in reactors for scale-up production) can be generated by operating methods for cell line generation under such and/or similar conditions or by testing clones under such desired conditions to identify those cell lines having the desired properties. In certain embodiments, cell lines additionally optimized for growth in media supplemented with compounds that generally retard or retard cell growth can be produced by selecting cells that exhibit normal or improved growth under such conditions as compared to most cells of the same type.

In certain embodiments, cells or cell lines expressing a protein of interest according to the invention can be used to produce a protein of interest. Included herein are methods of producing proteins using cells that have certain physiological properties that facilitate protein production and/or have been engineered to express one or more protein expression cofactors. In certain embodiments, cells that consistently and reproducibly express a protein of interest (e.g., for a period selected from the group consisting of: at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, and at least 9 months) as described herein are further modified and/or selected to provide an improved environment for protein production. In certain embodiments, cells engineered to express a protein of interest and a protein expressing a cofactor can be used to produce the protein of interest. In certain more specific embodiments, the protein of interest and/or the protein expression cofactor are consistently and reproducibly expressed.

In certain embodiments, the protein of interest is a biological product, such as an antibody for therapeutic use. Any protein of interest or fragment thereof can be produced according to the methods described herein, including but not limited to membrane proteins, transmembrane proteins, structural proteins, membrane anchoring proteins, cell surface receptors, secretory proteins, cytosolic proteins, heteromultimeric proteins, homomultimeric proteins, dimeric proteins, monomeric proteins, post-translationally modified proteins, glycosylated proteins, phosphorylated proteins, and proteolytically processed proteins. Specific examples of such proteins include, but are not limited to, antibodies (including antibody fragments such as Fab and Fab, Fab ', F (ab') ₂Fd, Fv, dAb, etc., single chain antibodies (scFv), single domain antibodies, heavy, light chains and all antibody classes, i.e., IgA, IgD, IgE, IgG and IgM), enzymes, coagulation factors, hormones, cellsFactors, ion channels, G-protein coupled receptors (GPCRs) and transporters. Other exemplary biologicals include those disclosed herein above.

In certain embodiments, a cell that facilitates protein production produces at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 250%, 500%, or at least 1000% more of a protein of interest than a reference cell. In certain embodiments, a cell that facilitates production of a protein produces a protein of interest that has at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 250%, 500%, or at least 1000% greater activity than the same protein of interest expressed in a reference cell. Activity may refer to, for example, enzymatic activity or binding activity or therapeutic activity of a binding partner to the protein of interest. In certain embodiments, a cell that facilitates production of a protein produces a protein of interest, wherein at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 250%, 500%, or at least 1000% more of the protein of interest is secreted relative to a reference cell. In certain embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or at least 99% of the protein of interest is located in a subcellular compartment in which the protein of interest is located in a cell that expresses the protein of interest without genetic engineering. Parameters to assess protein expression in a cell include quantification/analysis of protein production, processing, modification, intracellular localization or secretion, percentage of total protein optimally folded/processed. The reference cell may be a host cell from which the cell expressing the protein of interest is produced. Any cell disclosed herein can be used as a reference cell.

Proteins produced according to the methods provided herein can be characterized using methods known in the art, including protein sequencing, mass spectrometry, immunocytochemistry, methods of analyzing the degree or type of glycosylation and/or phosphorylation, and methods for determining covalent addition of chemical groups/or residues to the produced protein.

Cells of the greatest total amount of protein can be identified using methods known in the art (e.g., ELISA). In addition, assays that detect the activity of a protein or binding of a protein, such as activity assays, functional assays, binding assays (e.g., ELISA), secondary assays, animal studies, or cell-based assays, can be used to identify cells that result in the greatest percentage of active protein compared to the total production of protein.

In certain embodiments, a test protein is used to test the expression of a protein in a cell that expresses a protein of interest, wherein if the test protein is expressed at a level higher than that in a reference cell, the protein of interest is predicted to be expressed at a higher level in the same cell than in the reference cell. The test protein may include any protein, membrane protein, transmembrane protein, membrane anchoring protein, cell surface receptor, secretory protein, cytosolic protein, heteromultimeric protein, homomultimeric protein, dimeric protein, monomeric protein, post-translationally modified protein, glycosylated/phosphorylated/proteolytically processed protein, and any possible combination of the foregoing. In certain embodiments, a particular test protein may comprise an antibody, an ion channel, a GPCR, a transporter. Cells may be tested with only one or more test proteins. In the case of multiple test proteins, they may be of one or more types (e.g., multiple GPCRs, or only one GPCR, ion channel, or antibody).

Cells having physiological properties that are favorable for protein production (e.g., cells having increased endoplasmic reticulum quality) as well as cells that have been engineered for protein production (e.g., by introducing genes encoding proteins that are favorable for protein production) can be identified using methods known in the art, methods described herein, or combinations thereof. After such cells are identified, the cells can be cultured and cell lines stably expressing one or more genes encoding proteins useful for protein production can be generated according to the methods described herein. Such cell lines can be engineered to express the protein of interest by introducing a transgene before or after selecting for cells that are favorable for protein production, or by gene activation. Cells producing the protein of interest can then be identified using methods known in the art and/or described herein.

Cells having physiological properties that favor protein production include, but are not limited to, cells with increased viability, increased cell size, increased yield of endogenously expressed proteins, increased mitochondrial activity, increased stability and the ability to maintain properties against which they are selected, increased size of one or more organelles involved in protein processing (e.g., endoplasmic reticulum and ribosome), and increased content of one or more organelles involved in protein processing (e.g., lysosomes and endosomes). Such physiological properties can be compared to a reference cell or historical values for a reference cell.

FACS analysis in combination with standard methods of fluorescently labeled probes can be used to identify cells with physiological properties that favor protein production. In particular, markers of certain physiological properties are used to quantify the physiological property associated with protein expression and compare it to a reference cell. Such markers include molecules that detect cellular structures associated with protein expression such as ribosomes, mitochondria, ER, rER, golgi apparatus, TGN, vesicles, endosomes, and plasma membranes. Such a label may be a fluorescent dye. For example, the activity of certain organelles, such as mitochondria, can be assessed by measuring fluorescent metabolites. Fluorescent metabolites that report the activity of organelles/compartments, such as mitochondrial activity, or the integration of sugars onto proteins, which can be detected using, for example, fluorescent lectins, can be used. Protein markers specific for one or more organelles involved in protein processing can be expressed as fusion proteins with an autofluorescent protein tag such as Green Fluorescent Protein (GFP); and the membrane proteins and/or secreted proteins may be labeled with fluorescent probes. Probes (e.g., antibodies) to endogenously expressed proteins can be used as markers for organelle/compartment export. In particular, heteromultimeric membrane proteins are useful as a readout of protein expression activity in cells.

Without being bound by theory, an increase in fluorescence as measured by FACS may indicate that the cells have properties that are favorable for protein production, and such cells may be isolated for future use. In addition to such standard techniques, the methods described herein can also be used to identify cells that naturally have physiological properties that are favorable for protein production. For example, signaling probes complementary to target sequences of endoplasmic reticulum markers (e.g., ERp29, cytochrome P450, NADPH-cytochrome c reductase, calreticulin) can be used according to the methods described herein to identify cells with increased endoplasmic reticulum size. Cells that exhibit high fluorescence as measured by FACS may have increased ER size and/or content, and thus may have properties that facilitate protein production.

In one embodiment, cells having physiological properties that favor protein production are identified and then used to develop stable cell lines for production of the protein of interest. In another embodiment, cells are engineered to express a protein of interest, and cells expressing the protein of interest are then identified. Cells expressing the protein of interest and additionally having physiological properties favorable for protein production are then identified. Such cells are then used to develop stable cell lines that produce the protein of interest.

In an exemplary embodiment, a cell is transfected with a nucleic acid encoding a protein of interest; introducing into the cell a fluorescent oligonucleotide capable of detecting transcription of the nucleic acid; introducing into the cell a labeled fluorescent probe directed to a physiological property associated with protein expression; selecting cells that express the protein of interest and have an increased level of a physiological property associated with protein expression. Cell lines can then be established that consistently and reproducibly express the protein of interest.

Cells for use in the methods of protein production provided herein can be engineered to express a protein expression cofactor or a combination of two or more protein expression cofactors. In certain embodiments, the cell is engineered to express a splice variant, mutant, or fragment of a protein expression cofactor. In certain embodiments, the cell is engineered to express at least 2, 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, or 150 protein expression cofactors. Exemplary protein expression cofactors include: proteins that modulate Unfolded Protein Response (UPR) and genes encoding proteins that are modulated in UPR (e.g., ATF6 α (spliced), IRE1 α, IRE1 β, PERC Δ C, ATF4, YYI, NF-YA, NF-YB, NF-YC, XBP1 (spliced), EDEM 1); genes encoding proteins that shut down the apoptotic pathway induced by UPR (e.g., NRF2, HERP XIAP, GADD34, PPI α, PPI β, PPI γ, DNAJC 3); genes encoding proteins that affect cell growth, cell survival, cell death and cell size; b cell genes (e.g., blip-1, XBP1 (spliced)); genes encoding proteins involved in protein trafficking (e.g., Sec61P α, Sec61P β, Sec61P γ); a gene encoding a protein involved in glycosylation (e.g., SDF 2-L); genes encoding proteins involved in oxidation (e.g., ERO1 α, ER01 β; genes encoding anti-apoptotic proteins (e.g., Bcl-2sp, Bcl-xL, Bim trunk. mut., Ku70, 14-3-3q mut., VDAC2, BAP31 mut.); genes encoding proteins involved in endoplasmic reticulum-associated degradation (e.g., mannosidase 1, HRD 1); genes encoding proteins involved in calcium transport (e.g., STC1, STC2, SERCA1, SERCA2, COD 1); genes encoding proteins involved in lipogenesis/metabolism (e.g., INO1, PYC, SRP 1. DELTA. C, SREBP 2. C); and genes encoding proteins involved in folding and secretion of proteins (e.g., CRT (CaBP3), CNX, ERp57(PDIA3), BAP, ERd 3. DELTA. C), proteins involved in post-translational protein assembly into cell membranes (e.g., protein 573A 465), protein presentation in post-assembly, the cells are engineered to express any one or combination of genes involved in UPR. In another embodiment, the cell is engineered to express any one or combination of genes involved in UPR and at least one other gene encoding a protein known to be beneficial for protein production. Genes that modulate a UPR or are modulated in a UPR can be used; genes that alter cell growth, viability, apoptosis, cell death, cell size; genes encoding chaperones or factors involved in protein folding, assembly, membrane integration, cell surface presentation or secretion, post-translational modifications (including glycosylation/phosphorylation/proteolysis). In certain embodiments, the protein expresses a cofactor that alters a physiological characteristic of the cell.

Identification of cells expressing one or a combination of genes encoding proteins known to be beneficial for protein production can be accomplished using the methods described herein, e.g., a signaling probe can be generated that binds to a target sequence in the gene/mRNA of interest, which is then confirmed by FACS analysis for the presence of the gene/mRNA of interest.

In an exemplary embodiment, a cell is transfected with a first nucleic acid encoding a protein of interest and a second nucleic acid encoding a protein expression cofactor; introducing into the cell a fluorescent oligonucleotide capable of detecting transcription of the first nucleic acid and a fluorescent oligonucleotide capable of detecting transcription of the second nucleic acid; cells expressing the protein of interest and cells expressing the protein-expressing cofactor are selected. Cell lines can then be established that consistently and reproducibly express the protein of interest. The cell lines may be further tested for physiological properties associated with protein expression as described above.

In one embodiment, cells are first engineered to express protein expression cofactors; cell lines expressing the protein expression cofactors are established and cells of the cell lines are then engineered to express the protein of interest. In another embodiment, the cell is first engineered to express the protein of interest; cell lines expressing the protein of interest are established and cells of the cell lines are then engineered to express the protein expression helper factor. In another embodiment, the cells are engineered to express both the protein of interest and the protein expression cofactor. Cell lines that consistently and reproducibly express the protein of interest and/or protein expression cofactors can then be established as described herein.

In certain embodiments, a plurality of cells that have been engineered to express a protein of interest is provided. Such cells are then engineered to express protein expression cofactors and/or to select the cells from a plurality of cells that are most favorable for protein expression by using markers of physiological properties associated with protein expression as described above.

In certain embodiments, cell lines can be optimized for growth in media and/or suspension conditions free of animal components (as used in reactors for scale-up production) according to the methods provided herein. In certain more specific embodiments, the cell line can be optimized for growth in a medium comprising a growth-slowing component.

In other exemplary embodiments, methods for protein production are provided, comprising (i) identifying a cell having a physiological property that facilitates protein production; (ii) engineering the cell to express a protein of interest; (iii) generating a cell line stably expressing a protein of interest; (iv) culturing the cell under conditions suitable for production of the protein of interest; and (v) isolating the protein of interest.

In another embodiment, a method for protein production is provided, comprising (i) introducing into a cell at least one gene encoding a protein expression cofactor; (ii) identifying cells expressing the protein-expressing cofactor; (iii) engineering a cell to express a protein of interest; (iv) generating a cell line stably expressing a protein of interest; (v) culturing the cell under conditions suitable for production of the protein of interest; and (vi) isolating the protein of interest.

In another embodiment, a method for protein production is provided, comprising (i) introducing into a cell at least one gene encoding a protein expression cofactor; (ii) identifying cells expressing the protein-expressing cofactor; (iii) identifying cells having physiological properties that favor protein production; (iv) engineering a cell to express a protein of interest; (v) generating a cell line stably expressing a protein of interest; (vi) culturing the cell under conditions suitable for production of the protein of interest; and (vii) isolating the protein of interest. In one aspect of this embodiment, steps (ii) and (iii) are performed sequentially. In another aspect, steps (ii) and (iii) are performed simultaneously.

In another embodiment, a method for protein production is provided, comprising (i) introducing into a cell at least one gene encoding a protein expression cofactor and engineering the cell to express a protein of interest; (ii) identifying cells expressing the protein expression cofactor and the protein of interest; (iii) generating a cell line stably expressing the protein expression cofactor and the protein of interest; (iv) culturing the cell under conditions suitable for production of the protein of interest; and (v) isolating the protein of interest.

In another embodiment, a method for protein production is provided, the method comprising (i) introducing at least one gene encoding a protein expression cofactor into a cell and engineering the cell to express a protein of interest; (ii) identifying cells expressing the protein expression cofactor and the protein of interest; (iii) identifying cells having physiological properties that favor protein production; (iv) generating a cell line that consistently and reproducibly expresses both the protein expression helper factor and the protein of interest; (v) culturing the cell under conditions suitable for production of the protein of interest; and (vi) isolating the protein of interest. In one aspect of this embodiment, steps (ii) and (iii) are performed sequentially. In another aspect, steps (ii) and (iii) are performed simultaneously.

Host cells that can be used to produce cells suitable for protein production include primary cells and immortalized cells. In particular embodiments, the host cell can be, for example, a CHO cell, NS0 cell, PerC6 cell, yeast cell, insect cell, 293 cell, CACO cell, HUVEC, CHOK1, CHOKiSV, NS0, 293T cell, and insect cell.

In certain embodiments, the cell line of interest is or is capable of producing 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1 to 1.5, 1.5 to 2.0, 2.0 to 2.5, 2.5 to 3.0, 3.0 to 3.5, 3.5 to 4.0, 4.0 to 4.5, 4.5 to 5.0, 5.0 to 5.5, 5.5 to 6.0, 6.5 to 7.0, 7.0 to 7.5, 7.5 to 8.0, 8.0 to 8.5, 8.5 to 9.0, 9.0 to 9.5, 9.5 to 10.0, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 14 to 15, 15 to 16, 17 to 17, 17 to 19, 19 to 20, or more grams per liter of protein. In certain embodiments, the cells producing 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1 to 1.5, 1.5 to 2.0, 2.0 to 2.5, 2.5 to 3.0, 3.0 to 3.5, 3.5 to 4.0, 4.0 to 4.5, 4.5 to 5.0, 5.0 to 5.5, 5.5 to 6.0, 6.5 to 7.0, 7.0 to 7.5, 7.5 to 8.0, 8.0 to 8.5, 8.5 to 9.0, 9.0 to 9.5, 9.5 to 10.0, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 15 to 19, 17 to 25 or more grams per liter of cell line of interest are produced within 2, 3, 4, 15 to 3, 15, 17, or 9 months. In certain embodiments, the cell is stable for 1, 3, 4, 5, 6, 7, 8, 9, 10 months, wherein the level of protein does not change by more than 30%. In certain embodiments, the cells are stable in continuous culture for 1 year, 2 years, or longer, wherein the level of protein does not change by more than 30%. In certain embodiments, the protein of interest is a biological product or protein that can be used for clinical applications. In certain embodiments, the protein of interest is an antibody. In certain embodiments, the protein of interest is modified, post-translationally modified, or glycosylated. In certain embodiments, the protein produced by the cell is modified, post-translationally modified, or glycosylated with a second protein that the cell is engineered to comprise.

In certain embodiments, the present invention provides methods of producing an equivalent bioproduct product (e.g., a bioequivalent or biosimilar bioproduct) that matches the properties of an existing bioproduct product in a short period of time.

The matched panel of subjects of the invention may be generated by generating different cell lines for the panel sequentially, in parallel, or a combination of both. For example, each cell line can be prepared separately and subsequently matched. More preferably, to minimize differences between cell lines, sequentially produced cell lines may be frozen at the same stage or passage number and thawed in parallel. More preferably, the cell lines are prepared in parallel. In certain embodiments, the matched panel of subjects may be generated by generating each cell line of the panel using substantially the same conditions, protocols, or cell culture steps.

In a preferred embodiment, the cell lines in the subject panel are screened or assayed in parallel.

According to the present invention, the cell lines of the matched experimental group are maintained under the same cell culture conditions, including but not limited to the same medium, temperature, etc. All cell lines in the subject group are passaged at the same frequency, which may be any desired frequency, depending on a number of factors, including cell type, growth rate. As will be appreciated, in order to maintain a substantially equal number of cells from cell line to cell line in the subject group, the number of cells should be periodically normalized.

According to this method, the cells may be cultured in any cell culture format, provided that the cells or cell lines are dispersed in a single culture prior to the step of measuring the growth rate. For example, for convenience, cells may be initially combined for culture under desired conditions, and then individual cells separated into 1 per cell or vessel.

Cells may be cultured in multi-well tissue culture plates in any convenient cell number. Such plates are readily commercially available and are well known to those of ordinary skill in the art. In certain embodiments, the cells may be cultured, preferably in vials or in any other convenient form, various forms will be known to the skilled person and readily commercially available.

In embodiments that include a step of measuring the growth rate, the cells are cultured for a sufficient time to adapt them to the culture conditions before the growth rate is measured. As the skilled artisan will appreciate, the length of time will vary depending on a number of factors, such as the cell type, the conditions selected, the culture format, and can be any amount of time from 1 day to several days, 1 week, or more.

Preferably, each individual culture of the plurality of isolated cell cultures is maintained under substantially equivalent conditions, including a standardized maintenance schedule, discussed below. Another advantageous feature of this method is that a large number of individual cultures can be maintained simultaneously, allowing identification of cells with the desired trait group, even if very rare. For these and other reasons, according to the present invention, a plurality of isolated cell cultures are cultured using an automated cell culture method such that the conditions are substantially equivalent for each well. Automated cell culture prevents the inevitable variability inherent in artificial cell culture.

Any automated cell culture system may be used in the methods of the invention. Many automated cell culture systems are commercially available and well known to the skilled person. In certain embodiments, such systems may be suitable for use in the culture of a plurality of isolated cultures of automated or standardized cells or cell lines. In certain embodiments, such systems may be suitable for automating or standardizing the culture of multiple isolated cultures of cells or cell lines under substantially identical conditions. In certain embodiments, such systems may be suitable for use in parallel culture of multiple isolated cultures of automated or standardized cells or cell lines under substantially identical conditions. In certain embodiments, such systems may be suitable for automating or standardizing the culture of a plurality of isolated cultures of cells or cell lines such that at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 physiological properties of the cells are maintained during the culture. In certain embodiments, such systems may be suitable for use in the culture of multiple isolated cultures of automated or standardized cells or cell lines such that the cells or cell lines stably express the RNA or protein of interest. In certain embodiments, this is done A system-like system may be suitable for automating or standardizing the culture of a plurality of isolated cultures of cells or cell lines such that at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more than 50 physiological properties of the cells are maintained and such that the cells or cell lines stably express the RNA or protein of interest. In certain embodiments, the automated system is a robotic system. Preferably, the system includes independently moving channels, a multi-channel head (e.g., 96-point head) and a clamp or preferential pick arm, and a HEPA filtration device to maintain sterility during operation. The number of channels in the pipettor should be suitable for the culture format. A convenient pipette has, for example, 96 or 384 channels. Such systems are known and commercially available. For example, MICROLAB STAR^TMAn instrument (Hamilton) can be used in the process of the invention. An automated system should be able to perform a variety of desired cell culture tasks. Such tasks will be known to those of ordinary skill in the art. They include, but are not limited to: removing culture medium, replacing culture medium, adding reagents, washing cells, removing wash solution, adding dispersants, removing cells from culture vessels, adding cells to culture vessels, and the like.

The generation of the cells or cell lines of the invention may comprise a number of isolated cell cultures. However, the advantages provided by the method increase with increasing cell number. There is no theoretical upper limit on the number of cells or isolated cell cultures that can be utilized in the method. According to the present invention, the number of isolated cell cultures may be 2 or more, but more preferably is at least 3, 4, 5, 6, 7, 8, 9, 10 or more isolated cell cultures, such as at least 12, at least 15, at least 20, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 48, at least 50, at least 75, at least 96, at least 100, at least 200, at least 300, at least 384, at least 400, at least 500, at least 1000, at least 10,000, at least 100,000, at least 500,000 or more.

In certain embodiments, the cells and cell lines of the invention cultured as described are cells that have previously been selected positive for a nucleic acid of interest, which may be an introduced nucleic acid encoding all or part of a protein of interest, or an introduced nucleic acid that activates transcription of a sequence encoding all or part of a protein of interest. In certain embodiments, a cell cultured as described herein is one in which at least two have been selected positive for an RNA of interest or an RNA encoding a protein of interest.

For the preparation of the cells and cell lines of the invention, techniques such as those described in U.S. Pat. No. 6,692,965 and WO/2005/079462 may be used. Both of these documents are incorporated by reference herein in their entirety. This technique provides real-time evaluation of millions of cells, allowing any desired number of clones (from hundreds to thousands of clones). Using cell sorting techniques, such as flow cytometry cell sorting (e.g., with a FACS machine) or magnetic cell sorting (e.g., with a MACS machine), 1 cell/well can be automatically deposited in culture vessels (e.g., 96-well culture plates) with high statistical reliability. The speed and automation of the technology allows for easy isolation of multigene recombinant cell lines. In certain embodiments, cells that are positive for the desired signal (i.e., cells that express the desired RNA) are pooled. Such an assemblage may then be subjected to a second round of selection. In certain embodiments, the collection of cells is subjected to a total of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or at least 50 rounds of selection.

Using this technique, RNA sequences can be detected for a protein of interest using signaling probes, also known as molecular beacons or fluorescent probes. In certain embodiments, the vector comprising the coding sequence has additional sequences that encode an RNA tag sequence. By "tag sequence" is meant a nucleic acid sequence that is part of the expressed RNA or RNA to be detected by a signaling probe. The signaling probe can detect a variety of RNA sequences, any of which can be used as a tag, including those encoding peptides and protein tags described above. The signaling probe may be directed against the tag by designing the probe to include a portion complementary to the sequence of the tag. The tag sequence may be the 3' untranslated region of the plasmid, which is co-transcribed with the transcript of the protein of interest, and includes the target sequence for binding of the signaling probe. The tag sequence may be in-frame with the protein-coding portion of the messenger of the gene or out-of-frame with it, depending on whether it is desired to tag the protein produced. Thus, the target a large bamboo or straw hat with a concial crown, broard brim and handle sequence need not be translated for detection by a signaling probe. The tag sequence may comprise multiple target sequences, which may be the same or different, with one signaling probe hybridizing to each target sequence. The tag sequence may be located within the RNA encoding the gene of interest, or the tag sequence may be located within the 5 '-or 3' -untranslated region. The tag sequence may be an RNA having a secondary structure. The structure may be a three-arm connection structure. In certain embodiments, the signaling probe detects a sequence within the coding sequence of the protein of interest. The tag sequence may comprise chemically modified nucleotides. Tag sequences can be generated and used as described in international patent application publication No. wo2005/079462 (application No. pct/US05/005080), published on 9/1/2005.

After transfection of the DNA construct into cells and subsequent drug selection (if used), or after gene activation, molecular beacons (e.g., fluorescent probes) each targeting a different tag sequence and differentially labeled may be introduced into the cells, and a flow cytometry cell sorter used to isolate cells positive for their signal (multiple rounds of sorting may be performed). In one embodiment, the flow cytometry cell sorter is a FACS machine. MACS (magnetic cell sorting) or laser ablation of processed negative cells using laser activated analysis may also be used. Other fluorescence plate readers can also be used, including those compatible with high throughput screening. Signal positive cells take up at least one copy of one or more introduced sequences and may integrate it into their genome. Cells that introduce the messenger for the protein of interest are then identified. For example, a coding sequence may be integrated into a cell at a different location in the genome. The expression level of the introduced sequence may vary based on copy number or integration site. In addition, cells comprising the protein of interest can be obtained in which one or more of the introduced nucleic acids is episomal (episomal) or results from gene activation.

Signaling probes for use in the present invention are known in the art and are typically oligonucleotides comprising a sequence complementary to a target sequence and a signal emitting system arranged such that no signal is emitted when the probe is not bound to the target sequence and a signal is emitted when the probe is bound to the target sequence. By way of non-limiting example, a signaling probe can comprise a fluorophore and a quencher located on the probe such that the quencher and fluorophore are bound together on the unbound probe. When the probe binds to the target sequence, the quencher separates from the fluorescent group, resulting in emission of a signal. For example, International publication WO/2005/079462 describes a number of signaling probes that may be used in the generation of cells and cell lines of the present invention. The methods described above for introducing nucleic acids into cells can be used to introduce signaling probes.

Where a tag sequence is used, each vector (where multiple vectors are used) may comprise the same or different tag sequences. Regardless of whether the tag sequences are identical, the signaling probes may comprise different signaling emitters, e.g., fluorophores with different colors, etc., such that the expression of each subunit can be separately detected. By way of illustration, a signaling probe that specifically detects a first RNA of interest (e.g., an mRNA, siRNA, or RNA oligonucleotide) can comprise a red fluorophore, a probe that detects a second RNA of interest (e.g., an mRNA, siRNA, or RNA oligonucleotide) can comprise a green fluorophore, and a probe that detects a third RNA of interest (e.g., an mRNA, siRNA, or RNA oligonucleotide) can comprise a blue fluorophore. One of ordinary skill in the art will know of other methods for differentially detecting the expression of 3 subunits in triple transfected cells using signaling probes.

In one embodiment, the signaling probe is designed to be complementary to a portion of the RNA encoding the protein of interest or a portion of the 5 'or 3' untranslated region. Even if a signaling probe designed to recognize a messenger RNA of interest is capable of detecting falsely endogenously expressed target sequences, the ratio of these is such as to be compared to the ratio of the sequence of interest produced by the transfected cells, so that the sorter is able to distinguish between 2 cell types.

The expression level of the protein of interest may vary from cell to cell or from cell line to cell line. Expression levels in a cell or cell line can also decline over time due to epigenetic events such as DNA methylation as well as gene silencing and loss of transgene copies. These variations can be attributed to various factors such as the copy number of the transgene taken up by the cell, the site of genomic integration of the transgene, and the integrity of the transgene after genomic integration. Expression levels can be assessed using FACS or other cell sorting methods (i.e., MACS). Additional rounds of introducing signaling probes may be used, for example, to determine whether and to what extent cells remain positive over time for any one or more of the RNAs they were originally isolated.

Optionally, one or more replicate sets of cultures for one or more growth rate sets may be prepared. In some cases, it may be advantageous to freeze a replicate set of one or more growth frames, for example, to serve as a freezing stock. However, according to this method, the frozen cell stock can be prepared at any time as desired and at any and many points during its production. Methods for freezing cell cultures are well known to those of ordinary skill in the art. For example, the replicate group may be frozen at any temperature, for example at-70 ℃ to-80 ℃. In one embodiment, the cells are incubated until 70-100% confluence is reached. Next, the medium was aspirated and a solution of 90% FBS and 10% medium was added to the plate, isolated and frozen.

The present invention contemplates performing the method with any number of replicate groups using different culture conditions. That is, the method may be comprised of a first plurality (set) of isolated cell cultures cultured under a first set of culture conditions and a second set of isolated cell cultures cultured under a second set of conditions different from the first, and so on for any desired number of sets of conditions. Methods using different sets of conditions may be performed simultaneously or sequentially or a combination of both (e.g., 2 sets followed by 2 more sets at the same time, etc.).

One advantage of the methods described herein for selecting cells having consistent functional expression of a protein of interest is that the cells are selected by function rather than by the presence of a particular nucleic acid in the cell. A cell comprising a nucleic acid encoding a protein of interest may not express it, or even if the protein is produced, the protein may not be functional for a number of reasons, or have altered function compared to "native" function (i.e., function in the cell in its normal context of naturally expressed protein). By selecting cells based on function, the methods described herein enable the identification of novel functional forms. For example, multiple cells can be identified that have multiple degrees of function in the same assay, e.g., with the same test compound or with a series of compounds. The difference function provides a series of functional "properties". Such properties are e.g. used to identify compounds that differentially affect different functional forms of the protein. Such compounds are useful for identifying functional forms of proteins in particular tissues or disease states, and the like.

An additional advantage of the methods for producing the cells and cell lines of the invention, including cells expressing a complex protein or multiple proteins of interest, is that the cells can be produced in significantly less time than by conventional methods. For example, depending on a number of factors (including the number of cells required for a functional assay, the growth rate frame and whether it is complete, and other factors), cells expressing proteins that can be confirmed to have function can be produced in as little as 2 days or 1 week, but even 2 weeks, 3 weeks, 1 month, 2 months, 3 months, or even 6 months of production time is significantly faster than can be achieved by conventional methods, even for complex proteins or multiple proteins.

In another aspect, the invention provides methods of using the cells and cell lines of the invention. The cells and cell lines of the invention may be used in any application where a functional protein of interest is desired. The cells and cell lines can be used, for example, in vitro cell-based assays or in vivo assays in which cells are implanted into an animal (e.g.; non-human animal) to, for example, screen for modulators; producing proteins for crystallography and binding studies; and studying the effects of compound selectivity and dose, receptor/compound binding kinetics and stability, and receptor expression on cell physiology (e.g., electrophysiology, protein trafficking, protein folding, and protein regulation). The cells and cell lines of the invention can be used to examine the effect of proteins of interest in knock-down (knock-down) studies.

The cells and cell lines of the invention can also be used to identify soluble bioproduct competitors for functional assays, biopanning (e.g., using phage display libraries), gene chip studies to assess changes in gene expression, two-hybrid studies to identify protein-protein interactions, knock-down of specific subunits in cell lines to assess their effects, electrophysiology, studies of protein trafficking, studies of protein folding, studies of protein regulation, production of antibodies to proteins, isolation of probes to proteins, isolation of fluorescent probes to proteins, studies of the effect of expression of proteins on overall gene expression/processing, studies of the effect of expression of proteins on overall protein expression and processing, and studies of the effect of expression of proteins on cell structure, properties, study of the role of the features.

The cells and cell lines of the invention are further used to characterize proteins of interest (DNA, RNA or proteins) including DNA, RNA or protein stoichiometry, protein folding, assembly, membrane integration or surface presentation, conformation, activity state, activation potential, response, function and cell-based assay function, wherein the protein of interest comprises a multigene system, complex or pathway, whether all components of these are provided by one or more target genes introduced into the cell or any combination of introduced and endogenously expressed sequences.

Cells and cell lines that have been engineered to express one or more subunits of a multimeric (dimerized, trimerized, or higher multimerized) protein may produce different forms of the multimeric protein. The present invention provides methods for differentiating cells having different forms of multimeric proteins. The functional form of a multimeric protein may vary depending on the following factors: physiological state of the cell, alternative splicing or post-translational modification of the target (including proteolysis), its binding to accessory or interacting factors or its folding, assembly or integration in the cell membrane. The assembly and stoichiometry of the different subunits also increases the number of possible functional forms of the heteromultimeric target. The functional forms may differ in their response to the test compound or in their kinetics of activity over time. Comparative analysis of cells expressing different functional forms of multimeric proteins allows identification of cells comprising a particular functional form.

In certain embodiments, a multimeric protein is a physical or biochemical association of at least two protein subunits. In certain embodiments, a multimeric protein is a physical or biochemical association of a protein subunit with a cofactor for the protein subunit. In certain embodiments, the multimeric protein has at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 subunits. The subunits may be the same polypeptide or different polypeptides or a combination thereof. The multimeric protein may be any multimeric protein disclosed herein.

Functional activity or pharmacological properties as described herein of a cell line expressing a target of interest can be determined by testing the effect of one or more compounds on the activity of the target in the cell line. Grouping or classifying clones according to such pharmacological properties can be used to generate a panel of cell lines representing each possible form of the target. The representation of all possible functional forms can be sought by saturation screening, i.e. by testing at least a number of cell lines, such that each form determined by its pharmacological properties is represented by at least 2, 3, 5, 10, 25, 50 or at least 100 cell lines.

In certain embodiments, in the cell cycle Specific point (e.g., M, S, G)₁Or G₂Phase) to determine the effect of the compound on the target of interest. In another embodiment, the effect of a compound on a target of interest is monitored over time (e.g., within 1, 5, 10, 15, 20, 30, 40, 50 seconds; within 1, 5, 10, 15, 20, 30, 40, 50 minutes; within 1, 5, 10, 15, 20 hours; within 1, 2, 5, 10, 20, 30 days; or within 1, 2, 5, 10 months).

In certain embodiments, for heteromultimeric proteins, the pharmacological properties of a plurality of heteromultimeric proteins of interest, i.e., one property per cell line, can be produced using a plurality of cell lines each comprising all of the subunits. Differences in physiological properties between cell lines distinguish different forms of heteromeric proteins of interest, such as variable assembly or stoichiometry of subunits.

Comparison of cells expressing different or fewer than all subunits can be used to determine the functionality of individual subunits.

Examples of heteromultimeric proteins in which different combinations of subunits are available and can be functionally tested include, but are not limited to:

GABA (A) receptor heteromultimer combinations (see, e.g., Olsen and Sieghart, "International Union of pharmacology, LXX. Subtypes ofy-Aminobutyric Acid _AReceptors: classification of the Basis of SubunitCompsition, Pharmacology, and function. update ", Pharmacological reviews, 60: 243-260, 2008, which is incorporated herein by reference in its entirety):

GABA (a) subunits include, but are not limited to, GABRA1(α i), GABRA2(α 2), GABRA3(α 3), GABRA4(α 4), GABRA5(α 5), GABRA6(α 6), GABRB1(β 1), GABRB2(β 2), GABRB3(β 3), GABRG1(γ 1), GABRG2(γ 2), GABRG3(γ 3), GABRD (δ), GABRE (ε), GABRP (π), and GABRQ (θ);

GABA (a) subunit combinations include, but are not limited to, α 1 β 2 γ 2, α 2 β γ 2, α 3 β γ 2, α 4 β 2 δ, α 4 β 3 δ, α 5 β γ 2, α 6 β 2 δ, α 6 β 3 δ, ρ, α 1 β 3 γ 2, α 1 β δ, α 5 β 3 γ 2, α β l γ, α β 1 δ, α β, α 1 α 6 β γ, α 1 α 6 β δ, ρ 1, ρ 2, ρ 3, α β γ 1, α β γ 3, α β θ, α β ∈, and α β π;

nAChR heteromultimeric receptor combinations (see, e.g., Gotti, Zoli and Clementi, "Brain anionic acetylcholinergicide receptors: native subtypes and theirrelevance," Trends in Pharmacological Sciences, 27: 482-491, 2006, and N.Millar Neuropharmacology Vol.56, Issue 1, 2009, pp.237-246, the entire contents of which are incorporated herein by reference in their entirety):

nAChR subunits include, but are not limited to, CHRNA1(α 1), CHRNA2(α 2), CHRNA3(α 3), CHRNA4(α 4), CHRNA5(α 5), CHRNA6(α 6), CHRNA7(α 7), CHRNA8(α 8), CHRNA9(α 9), CHRNA10(α 10), CHRNB1(β 1), CHRNB2(β 2), CHRNB3(β 3), CHRNB4(β 4), CHRND (δ), and CHRNE (∈);

nAChR subunit combinations include, but are not limited to, α 1 β 1 γ δ, α 1 β 1 δ ∈, α 2 α 4 β 2, α 2 α 5 β 2, α 2 β 4, α 2 α 6 β 2, α 3 α 4 α 6 β 2, α 3 α 5 β 2, α 3 α 3 β 4, α 3 α 5 β 2 β 4, α 3 α 5 β 4, α 3 α 6 β 2, α 3 β 3, α 3 β 2 β 4, α 3 β 4, α 4 α 5 β 2, α 4 α 6 β 2 β 3, α 4 β 2, α 4 β 4, α 6 β 2 β 3, α 6 α 4 β 2, α 6 β 3 β 4, α 6 β 2 β 3 α 6 β 4, α 7 α 8, α 8 α 9 α 6 β 3 β 4, α 6 β 4, α 7 β 8 α 9 α 4, α 6 β 4;

5-HT₃heteromeric receptors (see, e.g., n.m. barnes et al, Neuropharmacology56 (2009)273-284, which is incorporated herein by reference in its entirety):

5-HT₃subunits include, but are not limited to, HTR3A (a), HTR3B (B), HTR3C (C), HTR3D (D), and HTR3E (E);

5-HT₃combinations of subunits include, but are not limited to, AB, AC, AD, and AE;

glycine heteromeric receptors (see, e.g., JW Lynch Neuropharmacology56(2009)303-309, which is incorporated herein by reference in its entirety):

Glycine receptor subunits include, but are not limited to, GLRA1(α 1), GLRA2(α 2), GLRA3(α 3), GLRA4(α 4), and GLRB (β);

combinations of glycine receptor subunits include, but are not limited to, α 1 α 3, α 1 β, α 2 β, α 3 β and α 4 β;

glutamate heteromeric receptors (see, e.g., perris, Neuropharmacology56(2009) 131-140; Jane, Neuropharmacology56(2009) 90-113; and w.lu, Neuron, 62, (2009)2, 254-268, which are incorporated herein by reference in their entirety):

glutamate receptor subunits include, but are not limited to, GRIK1(K1), GRIK2(K2), GRIK3(K3), GRIK5(K5), GRIM (A1), GRIA2(A2), and GRIA3 (A3);

glutamate receptor subunit combinations include, but are not limited to, K2K3, K1K2, K1K5, K2K5, A1A2, A1A3, and A2 A3;

ATP-gated P2X heteromeric receptors (see, e.g., M.F. Jarvis, B.S. Khakh, Neuropharmacology56(2009) 208-215; and S Robertson, Current opinion in Neurobiology 11, 2001, 378-386, which are incorporated herein by reference in their entirety):

ATP-gated P2X receptor subunits include, but are not limited to, P2RX1(X1), P2RX2(X2), P2RX3(X3), P2RX4(X4), P2RX5(X5), P2RX6(X6), and P2RX7 (X7);

ATP-gated P2X receptor subunit combinations include, but are not limited to, X1/X2, X1/X4, X1/X5, X2/X3, X2/X6, X4/X6, and X4/X7;

Taste receptors:

taste receptor subunits include, but are not limited to, TAS1R1(T1R1), TAS1R2(T1R2), TAS1R3(T1R 3);

taste receptor subunit combinations include, but are not limited to, T1R2/T1R3 (sweet taste receptor), T1R1/T1R3 (umami taste receptor);

GPCR heteromultimers, such as GPCR heterodimers (see, e.g., Prinser, Hague and Hall, "conjugation of G Protein-Coupled Receptors: Specificity and Functional Signal", pharmaceutical Reviews, 57: 289-298, 2005, the entire contents of which are incorporated herein by reference in their entirety), include, but are not limited to: HTR1B (5-HT1B)/HTR1D (5HT1D), ADORA1 (adenosine A1)/DRD1 (dopamine D1), ADORA1 (adenosine A1)/P2RY1(P2Y1), ADORA1 (adenosine A1)/GRM1(mGluR1{ alpha }), ADORA2 1 (adenosine A2 1)/DRD1 (dopamine D1), ADORA2 1 (adenosine)/GRM 1(mGluR 1), AGTR1 (angiotensin 1)/AGTR 1 (angiotensin 2), AGTR1 (angiotensin 1)/ADRB 1 ({ beta }2AR), AGTR1 (angiotensin 1)/BDKRB 1 (bradykinin B1), CASR (calcium sensitive receptor)/GRM 1 (calcium sensitive mGluR), CASR/CAMPR 1 (CAPR/CAPR 1)/CAPR 1 (CAPR 1/CAPR 1), CAPR 1/CAPR 1 (CAPR/CAPR 1), CAPR 1/CAPR 1 (CAPR/CAPR 1), CAPR 1/CAPR 36, DRD1 (dopamine D1)/DRD 1 (dopamine D1), DRD1 (dopamine D1)/SSTR 1, DRD1 (dopamine D1)/DRD 1 dopamine D1, EDNRA (endothelin A)/EDNRB (endothelin B), GABABR1/GABABR 1, MTNR1 1 (melatonin MT1)/MTNR1 1 (melatonin MT1), CHRM 1 (muscarinic M1)/CHRM 1 (muscarinic M1), OXTR (oxytocin)/AVPR 1 1 (vasopressin V1 1), OXTR (oxytocin)/SSTR 1 OPPR 1 (vasopressin V1), SOXTR (oxytocin)/AVPR 1 (vasopressin V1), S1PR1(S1P1)/S1PR 1(S1P 1)/OPPR 1 (ADOPPR 1/OPPR 1), ADOPPR 1 (ADOPPR 1/OPPR 72), ADOPPR 1/OPPR 1 (ADOPPR 1/OPPR 72), ADOPPR 1)/OPPR 1 (ADOPPR 1), ADOPPR 1/OPPR 1 (ADPR 1/OPPR 1), ADOPPR 1)/OPPR 1 (OPPR 1), ADOPPR 1 (OPPR 1/OPPR 1), ADOPPR 1 (OPPR 1)/OPPR 1 (OPPR 5 (OPPR 1/OPPR 5)/OPPR 1), and ADOPPR 1 (OPPR 1), and ADOPPR, ADRA1B (α 1BAR)/HRH1 (histamine H1), ADRA1B (α 1BAR)/ADRA1D (α 1DAR), ADRA1D (α 1DAR)/ADRB2D (β 2AR), ADRA2A (α 2AAR)/ADRB1(β 1AR), ADRA2A (α 2AAR)/OPRM1(μ -OPR), ADRB1(β 1AR)/ADRB2(β 2AR), ADRB2(β 2AR)/OPRD1(δ -OPR), ADRB2(β 2 AR)/OPRM 9 (K-OPR), ADRB2(β 2AR)/ADRB3(β 3AR), ADRB2(β 2AR)/M71-OR, OPRK1 (K-OPRK)/RD 7 (δ -OPRM) 8746 (OPRK)/3687458 (μ -OPRK 36R);

Voltage-gated calcium channel (CaV) multi-subunit complexes (see, e.g., WA Catterall et al Pharmacol Rev 200557411-425, and J Arikkath and K Campbell, Current Opinion in Neurobiology 2003, 13: 298-307, the entire contents of which are incorporated herein by reference in their entirety):

CaV subunits include, but are not limited to, CACNA1S (α)_1s)、CACNA1C(α_1c)、CACNA1D(α_1D)、CACNA1F(α_1F)、CACNA1A(α1_A)、CACNA1B(α_1B)、CACNA1E(α₁E) CACNB1(β 1), CACNB2(β 2), CACNB3(β 3), CACNB4(β 4), CACNA2D1(α 2 δ), CACNG1(γ 1), and CACNG2(γ 2);

CaV subunit combinations include, but are not limited to, alpha_1Sβ_1aα₂δγ₁、α_1Sβ_1aα₂δ、α_1Cβ₂α₂δγ、α_1Cβ₃α₂δγ、α_1Dβα₂δ、α_1Fβ₃α₂δ、α_1Fβ₂α₂δ、α_1Aβ₃α₂δ、α_1Aβ₄α₂δ、α_1Aβ₃α₂δγ₁、α_1Aβ₃α₂δγ₂、α_1Aβ₄α₂δγ₁、α_1Aβ₄α₂δγ₂、α_1Bβ₁α₂δ、α_1Bβ₃α₂δ、α_1Bβ₄α₂δ、α_1Bβ₃α₂δγ₂And alpha_1Eβα₂δ；

Voltage-gated sodium channel (NaV) multi-subunit complexes (see, e.g., WACatterall et al, pharmacol. rev., 200557397-409, which is incorporated herein by reference in its entirety):

NaV subunits include, but are not limited to, SCN1A (α 1), SCN2A (α 2), SCN3A (α 3), SCN4A (α 4), SCN5A (α 5), SCN8A (α 6), SCN9A (α 7), SCN1B (β 1), SCN2B (β 2), SCN3B (β 3), and SCN4B (β 4);

NaV subunit combinations include, but are not limited to, alpha₁/β₁、α₁/β₂、α₁/β₃、α₁/β₄、α₁/β₁/β₂、α₁/β₁/β₃、α1/β₁/β₄、α₁/β₂/β₃、α₁/β₂/β₄、α₁/β₃/β₄、α₂/β₁、α₂/β₂、α₂/β₃、α₂/β₄、α₂/β₁/β₂、α₂/β₁/β₃、α₂/β₁/β₄、α₂/β₂/β₃、α₂/β₂/β₄、α₂/β₃/β₄、α₃/β₁、α₃/β₃、α₄/β₁、α₅/β₁、α₅/β₂、α₅/β₃、α₅/β₄、α₅/β₁/β₂、α₅/β₁/β₃、α₅/β₁/β₄、α₅/β₂/β₃、α₅/β₂/β₄、α5/β₃/β₄、α₆/β₁、α₆/β₂、α₆/β₁/β₂、α₇/β₁、α₆/β₂2 and alpha₇/β₁/β₂；

An inward-rectifying potassium channel-heteromultimeric/multi-subunit complex (see, e.g., Y Kubo et al pharmacol. rev., 200557509-526, which is incorporated herein by reference in its entirety):

the inward rectifying potassium channel subunit includes but is not limited to KCNJ2 (K) _ir2.1)、KCNJ12(K_ir2.2)、KCNJ4(K_ir2.3)、KCNJ14(K_ir2.4)、KCNJ3(K_ir3.1)、KCNJ6(K_ir3.2)、KCNJ9(K_ir3.3)、KCNJ5(K_ir3.4) and KCNJ10 (K)_ir4.1)；

The inward-rectifying potassium channel subunit combinations include, but are not limited to, the combinations listed in the following table (table 1):

TABLE 1

Voltage-gated potassium channel-heteromeric/multi-subunit complexes (see, e.g., G Gutman et al pharmacol. rev., 200557473-508, which is incorporated herein by reference in its entirety):

voltage-gated potassium channel subunits include, but are not limited to, KCNA1 (K)_v1.1)、KCNA2(K_v1.2)、KCNA3(K_v1.3)、KCNA5(K_v1.5)、KCNA6(K_v1.6)、KCNA10(K_v1.8)、KCNB1(K_v2.1)KCNB2(K_v2.2)、KCNC4(K_V3.4)、KCND1(K_V4.1)、KCND2(K_v4.2)、KCND3(K_v4.3)、KCNF1(K_v5.1)KCNG1(K_V6.1)、KCNG2(K_v6.2)、KCNG3(K_V6.3)、KCNG4(K_v6.4)、KCNQ1(K_V7.1)、KCNQ2(K_v7.2)、KCNQ3(K_V7.3)、KCNQ4(K_v7.4)、KCNQ5(K_v7.5)、KCNV1(K_V8.1)、KCNV2(K_v8.2)、KCNS1(K_v9.1)、KCNS2(K_v9.2)、KCNS3(K_v9.3)、KCNH1(K_v10.1)、KCNH5(K_v10.2)、KCNH2(K_v11.1)、KCNH6(K_v11.2)、KCNH7(K_v11.3)、KCNAB1(K_vβ1)、KCNAB2(K_vβ2)、KCNAB3(K_vβ 3), KCNE1(minK), KCNE2(MiRP1), KCNE3(MiRP2), KCNE4(MiRP3), KCNE1L (KCNE 1-like), KCNIP1, KCNIP2, KCNIP3 and KCNIP 4;

voltage-gated potassium channel subunit combinations include, but are not limited to, the combinations listed in the following table (table 2):

TABLE 2

Calcium-activated potassium channel-heteromer and multi-subunit complexes (see, e.g., A Wei et al Pharmacol. Rev., 2005; 57: 463-472, which is incorporated herein by reference in its entirety):

calcium activated potassium channel subunits including but not limited to KCNMA1 (K)_Ca1.1)、KCNN1(K_Ca2.1)、KCNN2(K_Ca2.2)、KCNN3(K_Ca2.3)、KCNN4(K_Ca3.1)、KCNT1(K_Ca4.1)、KCNT2(K_Ca4.2) and KCNU1 (K)_Ca5.1)；

Calcium-activated potassium channel subunit combinations include, but are not limited to, the subunit combinations listed in the following table (table 3):

TABLE 3

Transient receptor potential channel-heteromeric/multi-subunit complexes (see, e.g., DEClapham et al Pharmacol. Rev., 2005; 57 (4): 427-450, which is incorporated herein by reference in its entirety):

Transient receptor potential channel subunits include, but are not limited to, TRPC1, TRPC3, TRPC4, TRPC5, TRPC6, TRPC7, TRPV-5, TRPM1, and TRPM 1-S;

transient receptor potential channel subunit combinations include, but are not limited to, those listed in the following table (table 4):

TABLE 4

Cyclic nucleotide-regulatory channel-heteromultimer/subunit complexes (see, e.g., FHofmann et al pharmacol. rev., 2005; 57 (4): 455-462, which is incorporated herein by reference in its entirety);

cyclic nucleotide-regulatory channel subunits including, but not limited to, CNGA, CNGB1a, CNGA2, CNGB1b, CNGA4, CNGA3, CNGB3, CNG4A, and CNGA 1;

cyclic nucleotide-regulatory channel subunit combinations include, but are not limited to, CNGA1/CNGB1a, CNGA2/CNGB1b/CNGA4, CNGA3/CNGB3, CNG4A/CNGA2/CNGB1b, CNGB1a/CNGA1, CNGB1b/CNGA2/CNGA4 and CNGB3/CNGA 3;

epithelial sodium channel/degenerate protein-heteromeric/multi-subunit complexes (see, e.g., AStaruschenko et al Biophys J, 2005; 88, 3966-3975, H Yamamura et al European J pharm, 2008, 600, 32-36, and S Kellenberger and L SchildPhysiol Rev, 2002, 82, 735-767, all of which are incorporated herein by reference in their entirety):

epithelial sodium channel/degenerate protein subunits include, but are not limited to, SCNN1A (ENaC α), SCNN1B (β), SCNN1G (γ), SCNN1D (ENaC δ), ACCN1, (ASIC2, two splice variants 2a and 2b), ACCN3(ASIC 3);

Epithelial sodium channel/degenerate protein subunit combinations include, but are not limited to, ENaC α β γ, ENaC α δ β γ, ENaC δ β γ, ASIC2a/ASIC2b, ASIC2a/ASIC3, and ASIC2b/ASIC 3.

For the heteromer proteins listed above and disclosed elsewhere herein, there may be many more combinations of subunits to be determined. Cell lines comprising the combinations listed above can be used as references to those cell lines that can express the novel combinations, thereby allowing the novel combinations to be determined. For example, different combinations of subunits can be characterized by the different response characteristics of different cell lines each expressing a set of the same subunits to a set of the same compounds. See, e.g., example 23, infra.

Large scale application of the method for heteromultimeric targets (e.g., GABA or acetylcholine ion channels) defined by a large gene family allows for cross-competitive analysis of multiple cell lines for all possible subunit combinations or subsets thereof.

In certain embodiments, gene activation is used in the methods, cells, and cell lines of the invention. Gene activation is described, for example, in International application publication WO 94/12650, which is incorporated by reference herein in its entirety. In certain embodiments, homologous recombination can be used to genetically modify cells encoding ENaC (epithelial sodium channel), GABA _A(gamma-aminobutyric acid type A), NaV (voltage gated sodium ion channel), sweet taste receptor, umami receptor, bitter taste receptor, CFTR (cystic fibrosis transmembrane conductance regulator) or GCC (guanylate cyclase C) receptors or receptor subunits of regulatory region, such that the genetic modification results in increased levels of EnaC, GABA and/or protein expression in the cell relative to the level of expression of the receptor or receptor subunit prior to the genetic modification_ANaV, sweet taste receptor, umami taste receptor, bitter taste receptor, CFTR or GCC receptor or receptor subunit expression. In certain embodiments, the genetic modification results in at least 10%, 50%, 100%, 500%, 1000%, 5000%, or at least 10,000% increase in expression. In certain embodiments, the cell expresses the receptor or receptor subunit at a background level prior to gene activation.

In certain embodiments, the promoter is introduced by homologous recombination with an ENaC, GABA endogenous to the cell_ANaV, sweet taste receptor, umami receptor, bitter taste receptor, CFTR or GCC receptor or one or more receptor subunits. The promoter may be a constitutively active promoter. In particular, the promoter may be one that is found in cells A constitutively active promoter. In other embodiments, the promoter is a conditional promoter. Cells useful in such methods have been described above. In certain embodiments, the promoter may be inserted randomly into the genome of the cell. The fluorescent oligonucleotide can then be used to select cells in which the randomly inserted promoter activates expression of the RNA of interest. In certain embodiments, the RNA of interest can be ENaC, GABA_ANaV, sweet taste receptor, umami taste receptor, bitter taste receptor, receptor for CFTR or GCC or RNA of one or more receptor subunits. In certain embodiments, regulatory DNA elements, such as enhancers or repressors, are inserted into the genome at random positions and cells are selected in which the RNA of interest is up-or down-regulated.

In certain embodiments, homologous recombination is used to introduce DNA into the genome of a cell, thereby allowing expression of an endogenous gene. In certain embodiments, the endogenous gene may be a gene encoding ENaC, GABA_ANaV, sweet taste receptor, umami taste receptor, bitter taste receptor, receptor for CFTR or GCC or one or more receptor subunits. In certain embodiments, the introduced DNA comprises a selectable marker, such as a gene encoding antibiotic resistance (e.g., DHFR). In certain embodiments, the endogenous gene is amplified in the genome of the cell. In certain embodiments, the endogenous gene is amplified such that the genome of the cell comprises 2 copies, 3 copies, 4 copies, 5 copies, 6 copies, 7 copies, 8 copies, 9 copies, 10 copies, at least 2 copies, at least 5 copies, at least 10 copies, at least 15 copies, at least 20 copies, or at least 25 copies of the endogenous gene. In certain embodiments, cells are selected for stable functional expression of the amplified gene.

Regulatory sequences can also be modified and expression achieved by the genetic variation methods described below. Genetic variability can be generated in cell lines, followed by selection for expression of ENaC, GABA using the fluorescent probes described herein_ANaV, sweet taste receptor, umami taste receptor, bitter taste receptor, receptor for CFTR or GCC or one or more receptor subunits.

In certain embodiments, the cell expresses a receptor or one or more receptor subunits of ENaC, GABAA, NaV, sweet taste receptor, umami taste receptor, bitter taste receptor, CFTR or GCC prior to application of gene activation or generation of genetic variability. The generation and selection of gene activation and/or genetic variability is then used to generate and select cells that express at least one additional subunit of another receptor or the same receptor and/or have a reduced expression level of the receptor or receptor subunit(s) that were expressed prior to application of gene activation or generation of genetic variability.

In certain embodiments, the cofactor is also expressed in the cell. The cofactor may be expressed in cells without genetic modification; the helper factor may be expressed as a transgene; or may express the cofactor through gene activation and/or genetic variability and selection as described above. A cofactor may be a factor that promotes transcription and/or translation and/or folding and/or subcellular localization and/or function of the receptor or receptor subunit of interest. Cofactors may facilitate the assembly of multi-subunit receptors.

In certain embodiments, provided herein are cell lines that express the complete receptor or 1, 2, 3, 4, or 5 subunits of a multi-subunit receptor of interest. The target multi-subunit receptor may be EnaC, GABA_ANaV, sweet taste receptor, umami taste receptor, or bitter taste receptor. In certain embodiments, none of the subunits of the receptor and the multi-subunit receptor of interest are expressed from the transgene. In certain embodiments, 1, 2, 3, 4, or 5 subunits of the multi-subunit receptor of interest are not expressed from a transgene. In certain embodiments, 1, 2, 3, 4, or 5 subunits of the complete receptor or multi-subunit receptor of interest are expressed from an amplified genomic region of the endogenous gene encoding the receptor or receptor subunit.

In certain embodiments, genomic DNA is transfected or microinjected into a population of cells, and cells expressing an RNA of interest are then selected. In certain embodiments, the RNA of interest can be EnaC, GABA_ANaV, a sweet taste receptor, an umami taste receptor or a bitter taste receptor or one or more subunits. The gene line DNA may be obtained from cells expressing the RNA of interest. In certain embodiments, the donor cell and the recipient cell of genomic DNA are from the same species. In certain embodiments, the donor cell and the recipient cell are from different species.

In certain embodiments, the donor cell of genomic DNA may be a cell of a human, mouse, insect, dog, donkey, horse, rat, guinea pig, bird or monkey. In certain embodiments, genomic DNA from 2, 3, 4, 5, 6, 7, 8, 9, or 10 different species is introduced. In certain more specific embodiments, different donor species of genomic DNA have orthologs of the gene of interest. In other embodiments, the donor species does not have an orthologue of the gene of interest.

In certain embodiments, the genomic DNA fragment is a defined region of the genome and comprises a gene of interest. In certain embodiments, the genomic DNA fragment is at least 1kb, 5kb, 10kb, 100kb, 500kb, or 1000kb in size.

Genomic DNA can be extracted from cells using any method known to those of ordinary skill in the art. An exemplary method for isolating genomic DNA is described in Short Protocols in molecular Biology, Ausubel et al (editor), John Wiley & Sons, Inc., 1999, Unit 2.2. In certain embodiments, the genomic DNA is treated very gently to avoid shearing of the DNA. In other embodiments, the genomic DNA is sheared to obtain smaller DNA fragments. In certain embodiments, the DNA is treated with a protease that does not contain dnase to remove any proteinaceous material from the DNA. In other embodiments, the genomic DNA is not treated with a protease, but instead care is taken not to disturb the proteins bound to the genomic DNA. In certain embodiments, the DNA is treated with an rnase enzyme that does not contain a dnase enzyme.

Genomic DNA can be introduced into cells using any method known to those of ordinary skill in the art. In certain embodiments, genomic DNA is transfected into a cell. In a more specific embodiment, lipofection is used to transfect genomic DNA into cells. Exemplary methods for introducing genomic DNA into cells are described in Short Protocols in molecular biology, Ausubel et al (editor), John Wiley & Sons, Inc., 1999, chapter 9.

The optimal amount of genomic DNA to be introduced into the cells can be determined by determining the number of cells expressing the RNA of interest. In certain embodiments, the amount of introduced gene DNA per cell corresponds to at least 1 genome equivalent, at least 10^-1Equivalent of at least 10 genomes^-2Equivalent of at least 10 genomes^-3Equivalent of at least 10 genomes^-4Equivalent of at least 10 genomes^-5Equivalent of at least 10 genomes^-6Equivalent of at least 10 genomes^-7The equivalent of one genome. In certain embodiments, the amount of genomic DNA introduced per cell corresponds to an equivalent of at most 1 genome, at most 10^-1Equivalent of one genome, at most 10^-2Equivalent of one genome, at most 10 ^-3Equivalent of one genome, at most 10^-4Equivalent of one genome, at most 10^-5Equivalent of one genome, at most 10^-6Equivalent of one genome, at most 10^-7The equivalent of one genome.

In certain embodiments, genomic DNA can be amplified by any method known to one of ordinary skill in the art. In certain more specific embodiments, the genomic DNA is amplified by whole gene amplification.

Any method can be used to identify and isolate those cells into which genomic DNA has been introduced. In certain embodiments, the DNA encoding the marker gene is introduced into the cell simultaneously with the genomic DNA. Cells positive for the marker gene also contain genomic DNA. Any marker gene known to those of ordinary skill in the art may be used. Illustrative examples of marker genes include genes whose gene products confer resistance to a particular antibiotic (e.g., neomycin resistance), genes whose gene products enable cells to grow on media lacking materials normally required for growth of the cell, or genes whose products encode visual markers. Visual markers that can be used in the methods of the invention are, for example, GFP. Cells into which DNA encoding a visual marker and genomic DNA have been introduced can be isolated using FACS.

In certain embodiments, microinjection is used to introduce genomic DNA into cells.

In certain embodiments, a fragment of genomic DNA is introduced into a vector to amplify the genomic DNA. Such vectors include, but are not limited to, phage, cosmids, or YACs. Any method known to those of ordinary skill in the art can be used to package and amplify genomic DNA.

In certain embodiments, provided herein are cell lines that express a multi-subunit receptor of interest at the same stoichiometric amount of subunits as those of the multi-subunit receptor of interest in a non-recombinant organism, wherein the cell lines are derived from cells that do not express the multi-subunit receptor of interest. In certain embodiments, provided herein are cell lines that express a receptor, including cell lines that express a multi-subunit receptor, wherein the pharmacological properties of the receptor in the cell line match the pharmacological properties of the receptor in cells that normally express the target in an organism. The receptor or multi-subunit receptor of interest may be, for example, ENaC, GABAA, NaV, sweet taste receptor, umami taste receptor, bitter taste receptor, CFTR or GCC. In certain embodiments, the cell line stably expresses the receptor or multi-subunit receptor in the absence of selective pressure as described above.

In certain aspects, the invention provides methods for identifying, isolating, and enriching cells that naturally occur and express an ENaC, GABAA, NaV, sweet taste receptor, umami receptor, bitter taste receptor, receptor of CFTR or GCC, or one or more receptor subunits. In certain embodiments, the naturally occurring cell naturally expresses a receptor or one or more receptor subunits of ENaC, GABAA, NaV, sweet taste receptor, umami receptor, bitter taste receptor, CFTR, or GCC.

In certain embodiments, the methods described herein rely on naturally occurring genetic variability and diversity. In certain embodiments, the isolated cells are represented by no more than 1/10, 1/100, 1/1000, 1/10,000, 1/100,000, 1/1,000,000, or 1/10,000,000 of the cells in the population. The population of cells may be primary cells harvested from an organism. In certain embodiments, genetic variability and diversity may also be increased using natural methods known to those of ordinary skill in the art. Any suitable method for generating or increasing genetic variability and/or diversity may be performed on the host cell. Without being bound by theory, the generation of such genetic variability produces modifications in the regulatory regions of genes encoding receptors or receptor subunits, such as ENaC, GABAA, NaV, sweet taste receptors, umami receptors, bitter taste receptors, CFTR or GCC. Cells expressing such subunits can then be selected as described above. In certain embodiments, cells endogenously expressing the protein of interest are isolated from clonal cells that have undergone at least 50, 100, 500, 750, or at least 1000 passages or have undergone continuous culture for at least 1, 2, 5, or 10 months or at least 1, 2, 5, 10 years.

In other embodiments, genetic variability may be obtained by exposing cells to ultraviolet light and/or X-rays (e.g., gamma rays). Genetic variability may also be obtained in a population of cells by introducing genomic DNA, cDNA and/or mRNA into the cells of the population. In particular embodiments, fragments of genomic DNA are introduced as described above. In a particular embodiment, a library of genomic DNA is introduced. In a particular embodiment, a cDNA library is introduced. In a particular embodiment, an expression DNA library is introduced. In certain embodiments, the genomic DNA, cDNA, and/or mRNA is derived from a cell type that is different from the cell type of the recipient cell. In particular embodiments, the genomic DNA, cDNA, and/or mRNA is derived from a taste cell.

In other embodiments, genetic variability may be obtained by contacting cells with EMS (ethyl methane sulfonate). In certain embodiments, genetic variability may be obtained by contacting cells with mutagens, carcinogens, or chemical agents. Non-limiting examples of such agents include deaminating agents such as nitrous acid, intercalating agents, and alkylating agents. Other non-limiting examples of such agents include bromine, sodium azide, and benzene.

In particular embodiments, genetic variability may be obtained by contacting the cells with suboptimal growth conditions such as hypoxia, low nutrients, oxidative stress or low nitrogen.

In certain embodiments, enzymes that cause DNA damage or reduce the fidelity of DNA replication or repair (e.g., mismatch repair) may be used to increase genetic variability. In certain embodiments, inhibitors of enzymes involved in DNA repair are used. In certain embodiments, compounds are used that reduce the fidelity of enzymes involved in DNA replication. In certain embodiments, proteins that cause DNA damage and/or reduce the fidelity of DNA replication or repair are introduced into cells (co-expression, injection, transfection, electroporation).

The duration of contact with certain conditions or agents depends on the conditions or agents used. In certain embodiments, seconds or minutes of contact are sufficient. In other embodiments, contact for hours, days, or months is necessary. One of ordinary skill in the art will know the duration and intensity of conditions that can be used.

Without being bound by theory, the method of increasing genetic variability produces a mutation or change in a promoter region of a gene, wherein the mutation or change results in a change in transcriptional regulation of the gene, such as gene activation, wherein the gene is more highly expressed than a gene with an unaltered promoter region. In certain embodiments, the gene encodes a receptor or receptor subunit of ENaC, GABAA, NaV, sweet taste receptor, umami receptor, bitter taste receptor, CFTR, or GCC. Typically, a promoter region comprises the genomic DNA sequence upstream of the transcription initiation site that regulates gene transcription, and may include a minimal promoter and/or enhancer and/or repression region. The promoter region may range from about 20 base pairs (bp) to about 10,000 bp or more. In particular embodiments, the method of increasing gene variability produces a mutation or alteration in an intron of a gene of interest that results in a change in the transcriptional regulation of the gene, such as gene activation, wherein the gene is expressed to a higher degree than a gene with an unaltered intron. In certain embodiments, the non-transcribed genomic DNA is modified. For example, promoters, enhancers or modifiers or repressing regions may be added, deleted or modified. In these cases, transcription of the transcript under control of the modified regulatory region can be used as a readout. For example, if a repressor is deleted, the transcripts of the gene inhibited by the repressor are tested for increased transcription levels.

In certain embodiments, the genome of a cell or organism can be mutated by site-directed mutagenesis or homologous recombination. In certain embodiments, oligonucleotide or triple helix mediated recombination may be used. See, e.g., Faruqi et al, 2000, Molecular and Cellular Biology 20: 990-1000 and Schleifman et al 2008, Methods Molecular Biology 435: 175-90.

In certain embodiments, fluorescent oligonucleotide probes or molecular beacons can be used to select cells in which the genetic modification is successful, i.e., cells in which the transgene or gene of interest is expressed. To identify cells in which a mutagenic or homologous recombination event is successful, fluorescent oligonucleotides that specifically hybridize to the mutagenic or recombinant transcript can be used. In certain embodiments, where cells are selected that endogenously express the protein of interest and are cells of a cell type that do not express the protein of interest, fluorescent oligonucleotides that specifically hybridize to RNA encoding the protein of interest can be used to isolate cells that express the desired protein. Isolation of the cells can be performed as described in U.S. Pat. No.6,692,965 to Shekdar et al, published at 2/17 2004 and International application No. PCT/US2005/005080 published as WO/2005/079462. In certain embodiments, cells that are positive for the desired signal (i.e., cells that express the desired RNA) are pooled. Such an assemblage may then be subjected to a second round of selection. In certain embodiments, the collection of cells is subjected to a total of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or at least 50 rounds of selection.

Cells and cell lines engineered to express multimeric proteins may comprise a plurality of different functional forms of the multimeric protein. The same cell or cell line engineered to comprise the multimeric-protein-complex may comprise different functional forms or different proportions of the same or different functional forms when cultured under different cell culture conditions. Culture conditions may vary in temperature, nutrient concentration, serum addition, ion concentration, cell density, synchronization of the cell cycle, pH, acidity, carbonation, atmospheric conditions, percentage of carbon dioxide, shaking, stirring, uv light exposure, metabolites, serum, amino acids, sugars, carbohydrates, proteins, lipids, detergents, growth factors, cofactors, vitamins, mutagens, chemicals, compounds, or trace metals activity or concentration. Different cells or cell lines engineered to comprise the same target may comprise different functional forms or different proportions of the same or different functional forms. The described methods can be used for comparative analysis of cells or cell lines engineered to contain the same or different multimeric proteins, cultured under the same or different cell culture conditions.

In certain embodiments, the different cells or cell lines used in the methods for characterizing multimeric proteins and producing cells or cell lines expressing different forms of a multimeric protein of interest are the cells or cell lines described herein, e.g., cells or cell lines having substantially identical physiological properties, cells or cell lines expressing a protein of interest that does not comprise a protein tag, or cells or cell lines having a Z' factor of at least 0.4 in a functional assay, or cells or cell lines cultured in the absence of selective pressure, or any combination thereof. In certain embodiments, the different cells or cell lines to be used are maintained in parallel under substantially the same culture conditions. The robotic methods described herein can be used to maintain and manipulate cells or cell lines that express different forms of a multimeric protein of interest. In certain embodiments, the invention provides a cell or cell line that expresses a multimeric (dimeric, trimeric or higher order multimerization) protein of interest from an introduced nucleic acid encoding said multimeric protein of interest or one or more subunits thereof, wherein the cell is cultured in the absence of selective pressure and/or wherein the protein is consistently expressed as described herein.

In certain embodiments, the invention provides a subject panel method of generating a cell line comprising a plurality of cell lines, wherein each cell line of the plurality of cell lines synthesizes a different form of a multimeric protein of interest. Multimeric proteins of interest are further disclosed above. Without being bound by theory, the different forms of multimeric proteins differ, for example, in their subunit combinations, post-translational modifications of the individual subunits, and/or subcellular localization (e.g., relative to the cytoskeleton; for transmembrane proteins: membrane integration/localization, exit endoplasmic reticulum, exit golgi apparatus; and for secreted proteins: exit endoplasmic reticulum, exit golgi apparatus). In certain embodiments, such a panel of subjects can be produced by starting with a particular clone or line of host cells, and then engineering the host cells to express one or more subunits of the multimeric protein of interest. In certain embodiments, a transgene encoding one or more subunits is introduced. In other embodiments, gene activation is utilized. Cells that have been successfully engineered to express one or more subunits can be selected by any method known to one of ordinary skill in the art. In certain embodiments, positive cells are selected using fluorescent oligonucleotides or molecular beacons (see, e.g., International application No. PCT/US2005/005080 published as WO/2005/079462). Cell lines were established from the identified cells. In certain embodiments, the resulting cell line is produced from the same material (e.g., the same host parent clone, the same nucleic acid used to engineer expression of one or more subunits) and the same protocol (e.g., the same cell culture method, the same genetic engineering method). Without being bound by theory, the resulting cell lines may vary in the insertion site of any transgene in the genome of the host cell and/or in the number of flying copies of any transgene in the genome. Surprisingly, it was found that the resulting cell lines can synthesize different forms of multimeric proteins. Cell lines that synthesize different forms of multimeric proteins can be identified by establishing the pharmacological properties of each cell line. Certain compounds were tested for their effect on multimeric proteins in various cell lines. Activity can be monitored over time. A dose-response curve can be established. In certain embodiments, at least 2, 5, 10, 25, 50, 100, 250, 500, 750, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 7500, or at least 10000 different compounds per cell line are tested to generate a pharmacological fingerprint of multimeric proteins synthesized by a particular cell line. The resulting pharmacological fingerprints of each cell line were then compared to each other. If the pharmacological fingerprints are substantially identical, then the form of the multimeric protein in the cell line is identical. If the pharmacological fingerprints are substantially different, the form of the multimeric protein in such cell lines is different.

In certain embodiments, if the reaction properties of the multimeric protein are the same for each compound (e.g., activated, inhibited, or not acting), then the forms are the same.

In other embodiments, the form is the same if the nature of the reaction is the same within the bounds of 0%, 25%, 10%, 5%, 1%, or 0.5% for each compound.

In certain embodiments, if the nature of the reaction of the multimeric protein is different (e.g., activated, inhibited, or not acting) for at least one compound, then the format is not the same. In certain embodiments, the format is different if the nature of the reaction of the multimeric protein is different (e.g., activated, inhibited, or not acting) for at least 50%, 25%, 10%, 5%, 1%, or 0.5% of the compounds tested. In other embodiments, the forms are different if the amount reacted for at least 1 compound is not within the bounds of at least 50%, 25%, 10%, 5%, 1%, or 0.5%. In other embodiments, the format is different if the amount reacted for at least 50%, 25%, 10%, 5%, 1%, or 0.5% of the test compound is not within the bounds of at least 50%, 25%, 10%, 5%, 1%, or 0.5%. Any algorithm known to those of ordinary skill in the art can be used to quantify and compare the pharmacological fingerprints of multimeric proteins in different cell lines.

In certain embodiments, the combination of biologically active multimeric proteins of interest in cells co-expressing the first subunit and the second subunit can be classified by comparing the pharmacological properties of the first subunit, the pharmacological properties of the second subunit, and the pharmacological properties of the mixed subunits. The first subunit pharmacological property can include a measurement that represents the effect of the compound on the biological activity of the multimeric protein as it is expressed in a cell that expresses the first subunit but not the second subunit of the multimeric protein. The second subunit pharmacological property can include a measure indicative of the effect of the compound on the biological activity of the multimeric protein as it is expressed in a cell that expresses the second subunit of the multimeric protein but does not express the first subunit. The mixed subunit pharmacological property can include a measure indicative of the effect of the compound on the biological activity of the multimeric protein as it is expressed in a cell expressing the first and second subunits of the multimeric protein. In certain embodiments, a biologically active multimeric protein of interest can be classified as (i) a homodimer of a first subunit if the first subunit pharmacological property has high similarity to that of a mixed subunit pharmacological property and the second subunit pharmacological property has low similarity to that of the mixed subunit; (ii) a homodimer of a second subunit if the second subunit pharmacological profile has high similarity to the mixed subunit pharmacological profile and the first subunit pharmacological profile has low similarity to the mixed subunit pharmacological profile; (iii) a heterodimer of a first subunit and a second subunit if the first subunit pharmacological profile has low similarity to the mixed subunit pharmacological profile and the second subunit pharmacological profile has low similarity to the mixed subunit pharmacological profile; or (iv) a combination of a homodimer of a first subunit and a homodimer of a second subunit if the mixed subunit pharmacological property is a combination of the pharmacological properties of the first subunit and the pharmacological properties of the second subunit.

In another embodiment, the composition of the biologically active multimeric protein of interest in cells co-expressing the first subunit and the second subunit can be classified by comparing the pharmacological profile of the cell of interest with the pharmacological profile of the first subunit, the pharmacological profile of the second subunit, and the pharmacological profile of the mixed subunits. In certain embodiments, a biologically active multimeric protein of interest may be classified as (i) a homodimer of a first subunit if the first pharmacological property has low similarity to the pharmacological property of the second subunit and high similarity to both the pharmacological property of the first subunit and the pharmacological property of the mixed subunit; (ii) a homodimer of a second subunit if said first pharmacological property has low similarity to said first subunit pharmacological property and high similarity to both said second subunit pharmacological property and said mixed subunit pharmacological property; (iii) a heterodimer of a first subunit and a second subunit if the first pharmacological property has low similarity to the first subunit pharmacological property, low similarity to the second subunit pharmacological property and low similarity to the mixed subunit pharmacological property; or (iv) a combination of a homodimer of a first subunit and a homodimer of a second subunit if the first pharmacological property has high similarity to the mixed subunit pharmacological property and the first pharmacological property is a combination of the first subunit pharmacological property and the second subunit pharmacological property.

In certain embodiments, the effect of the subunit on the biological activity of the multimeric protein in the cell can be characterized by comparing the pharmacological property from a cell expressing the subunit of interest to the pharmacological property from a cell not expressing the subunit of interest. In certain embodiments, the test pharmacological profile can be compared to a base pharmacological profile to characterize the effect. In the above embodiments, testing for a pharmacological property may comprise measuring a measure indicative of the effect of the compound on the multimeric protein having biological activity in a cell expressing the combination of pre-selected subunits and further expressing the subunit of interest; the basic pharmacological property may comprise a measure indicative of the effect of the compound on the multimeric protein having biological activity in a cell expressing the combination of preselected subunits but not the subunit of interest. The subunit of interest is not one of the subunits of the combination of pre-selected subunits. A subunit of interest can be characterized as having an effect on the biological activity of the multimeric protein if the test pharmacological property has a low similarity to the underlying pharmacological property, or as having no effect on the biological activity of the multimeric protein if the test pharmacological property has a high similarity to the underlying pharmacological property.

The pharmacological properties may be compared by calculating correlations between the pharmacological properties, such as, but not limited to, calculating similarity measures between the pharmacological properties.

In certain embodiments, two pharmacological properties are considered related if one of the pharmacological properties is measured within about 2%, about 5%, about 8%, about 10%, about 12%, about 15%, about 20%, about 25%, about 30%, or about 35% of the other pharmacological property.

In certain embodiments, two pharmacological properties may be considered to have a high similarity to each other if the measure of similarity calculated between them is above a predetermined threshold, or may be considered to have a low similarity to each other if the measure of similarity calculated between them is below a predetermined threshold. In certain embodiments, the predetermined threshold may be determined as a value representing a measure of similarity of one of the pharmacological properties measured to within about 2%, about 5%, about 8%, about 10%, about 12%, about 15%, about 20%, about 25%, about 30%, or about 35% of the other pharmacological property measured.

In certain embodiments, the pharmacological properties of a compound of interest may be expressed as a vector p,

p＝[p₁，...p_i，...p_n]

wherein p is _iIs a measurement of the ith constituent, e.g.The effect of the compound on the ith biological activity of a cell expressing a subunit of interest of the multimeric protein. In certain embodiments, n is greater than 2, greater than 10, greater than 100, greater than 200, greater than 500, greater than 1000, greater than 2000, greater than 2500, greater than 7500, greater than 10,000, greater than 20,000, greater than 25,000, or greater than 35,000. Each pharmacological property may also be represented as a vector p. In calculating the correlation, for each component i 1.. n, the measured quantity of the ith component of the vector representing the pharmacological property of the compound of interest may be compared to the measured quantity of the ith component of the corresponding vector representing the pharmacological property. However, there are many ways in which correlations can be calculated. In fact, any statistical method known in the art for determining the probability of correlation of two data sets can be used to identify whether a correlation exists between a pharmacological property and a pharmacological property of a compound of interest according to the methods of the present invention. For example, a similarity measure sim (pi) may be used₁，pi₂) Calculating the pharmacological Properties (pi) of the Compound of interest₁) With various pharmacological properties (pi)₂) The correlation between them. One way to compute the similarity measure sim (pi1, pi2) is to compute the euclidean distance to the negative 2 th power. In alternative embodiments, sim (pi1, pi2) may be calculated using measures other than euclidean distance, such as manhattan distance, chebyshev distance, vector angle, correlation distance, normalized euclidean distance, mahalanobis distance, squared pearson correlation coefficient, or minkowski distance. In certain embodiments, the pearson correlation coefficient, squared euclidean distance, squared euclidean sum or squared pearson correlation coefficient is used to determine similarity. Such measures can be calculated, for example, using AS (Statistical analysis systems Institute, Cary, North Carolina) or S-Plus (Statistical Sciences, Inc., Seattle, Washington). The use of such measures is described in Draghici, 2003, Data Analysis Tools for DNA microarray, Chapman &Hall, CRC Press London, Chapter 11, which is incorporated herein by reference in its entirety.

The correlation may also be calculated based on rank, where x_iAnd y_iIs a measured value arranged in ascending or descending numerical order, or the likeAnd (4) stages. See, e.g., Conover, practical Nonparametric statics, 2 nd edition, Wiley, (1971). Shannon mutual information may also be used as a similarity measure. See, for example, Pierce, An Introduction To information theory: symbol, Signals, and Noise, Dover, (1980), which is incorporated herein by reference in its entirety.

Some embodiments of the invention provide computer program products including any or all of the program modules shown in fig. 2. Aspects of the program modules are described further below.

Any assay known to those of ordinary skill in the art can be used to determine the activity of the multimeric protein of interest, thereby establishing a pharmacological fingerprint. Embodiments of the program module are described further below. Any assay used in the methods of the invention can be performed in a high throughput format.

In certain embodiments, the subject panels of the invention may be used in HTS or in combination with pharmaceutical chemistry to test and identify compounds that are selective for one or a set of targets; in particular, to identify compounds whose activity is specific for a particular form of multimeric protein of interest or which have a particular activity pattern or activity profile. Secondary assays, including in vivo tests using compounds, can be used to determine the presence, effect and function of the corresponding target in vivo, or its correlation as a biomarker (the assay methods are diverse and include brain imaging studies, animal studies, binding studies, behavioral patterns, PET scans, NMRI, etc.).

The present invention provides compositions for characterizing multimeric proteins. Without being bound by theory, a multimeric protein characterized by a functional criterion may reflect a particular stoichiometry or a combination or level of different particular stoichiometries of the multimeric protein. In certain embodiments, the multimeric protein is expressed in a cell that has been engineered to express the multimeric protein.

In certain embodiments, the composition of the dimer is determined using the methods of the invention.

In certain embodiments, the methods of the invention allow for the determination that dimers in a particular cell are a) homodimers of the first subunit; b) a homodimer of a second subunit; or c) a heterodimer of the first and second subunits. For example, such a method may comprise the steps of:

step A) the activity of the first subunit is tested in cells not expressing the second subunit.

Step B) testing the activity of the second subunit in cells not expressing the first subunit.

Step C) testing the activity of the first subunit and the second subunit in cells expressing the first and second subunits.

Comparing the activities obtained in steps A, B and C, and then inferring the combination of dimeric proteins in the cells expressing the first and second subunits. If the activity determined in step A is equal to the activity determined in step C, then the dimers formed in the cells co-expressing the first and second subunits are homodimers of the first subunit. If the activity determined in step B is equal to the activity determined in step C, then the dimers formed in the cells co-expressing the first and second subunits are homodimers of the second subunit. If the activity determined in step A and the activity determined in step B are both different from the activity determined in step C, then the dimer formed in the cells co-expressing the first and second subunits is a heterodimer of the first and second subunits. If the activity determined in step C is a combination of the activities observed in step A and step B, then the cells co-expressing the first and second subunits produce homodimers of the first subunit and homodimers of the second subunit.

In certain embodiments, the activity profile of the compound is measured over time.

Similar steps can be taken to determine subunit combinations of trimers and other multimeric proteins with higher order multimerization. In certain embodiments, the invention provides a panel of subjects comprising a plurality of cell lines, wherein each of the plurality of cell lines has been engineered to express the same subunit of a multimeric protein using the same protocol and the same host cell, wherein the multimeric protein produced differs between cell lines. The differences between multimeric proteins may be in: different combinations of subunits, different subunit stoichiometries, different post-translational modifications (including proteolysis), and/or different splicing of one or more subunits. Without being bound by theory, differences in multimeric proteins between cell lines may result, for example, from different insertion positions or copy numbers of subunits of introduced sequences introduced into the cells. In certain embodiments, the multimeric protein is characterized by measuring the production of a pharmacological property of at least 2, 5, 10, 50, 100, 250, 500, 1000, 2000, 5000, or at least 10,000 compounds on the activity of the multimeric protein. Such pharmacological properties of each cell line were compared with each other. If the pharmacological properties are the same, it is predicted that the composition of the multimeric protein is the same. If the pharmacological properties are different, it is predicted that the composition of the multimeric protein will be different.

In certain embodiments, the invention provides a subject panel of cell lines, wherein each subject panel comprises a plurality of cell lines, each of which expresses a different multimeric protein defined by the different pharmacological properties described above, wherein the different cell lines are produced using the same protocol and the same host cell. In certain embodiments, substantially the same cell culture protocol may be used. In certain embodiments, cell lines in a subject group may be treated in parallel or at different times but using identical or similar cells, conditions, or protocols.

In certain embodiments, the methods can be used for clones produced from different host cell lines. In certain embodiments, the host cell lines differ in their gene expression profiles. In such embodiments, different cell lines can be used to characterize the effect of a particular cofactor or endogenously provided factor on the formation of certain forms of the multimeric protein of interest.

In particular embodiments, the activity of a multimeric protein may be tested by contacting a cell expressing the multimeric protein with a compound that activates or inhibits the multimeric protein.

In certain embodiments, the activity profile of the multimeric protein is determined under certain conditions. Without being bound by theory, it is believed that the same conditions may have different effects on different forms of multimeric proteins. For example, their effect on multimeric proteins is tested against a number of different conditions. Exemplary conditions include: temperature, ion concentration in cell culture Medium, CO₂Concentration, cell density, synchronization of cell cycle, pH, acidity, carbonation, atmospheric conditions, percentage of carbon dioxide, shaking, stirring, uv light exposure, metabolites, nutrients, serum, amino acids, sugars, carbohydrates, proteins, lipids, detergents, growth factors, cofactors, vitamins, mutagens, chemicals, compounds, or trace metal activity or concentration. The activity profile can then be used to infer the composition of the multimeric protein.

In certain embodiments, the pharmacological properties of the multimeric protein are determined. For example, their effect on multimeric proteins is tested against a number of different compounds. Exemplary compounds include compounds known to modulate proteins or proteins of the same class or family, compounds known to have side effects in clinical studies, compounds with clinical efficacy, compounds that can have pharmacological activity, compounds of combinatorial chemical libraries, compounds, synthetic compounds, natural compounds, peptides, lipids, detergents, mutagens, fluorescent compounds, or polymers. The pharmacological properties can then be used to infer the composition of the multimeric protein.

The present invention enables the generation of a variety of cell lines expressing a protein of interest. The clonal cell lines of the present invention will have different absolute and relative levels of such expression. Large groups of such clones can be screened for activity using a number of known reference compounds. In this way, each isolated cell line will have a "fingerprint" of the response to the test compound, which represents the activity of differential functional expression of the protein. Cell lines can then be grouped based on the similarity of such responses to compounds. At least one cell line representing each functionally distinct expression profile may be selected for further study. The collection of these cell lines can then be used to screen a large number of compounds. In this way, compounds that selectively modulate one or more of the corresponding unique functional forms of a protein can be identified. These modulators can then be tested in secondary assays or in vivo models to determine what shows activity in these assays or models. In this context, modulators will be used as reference compounds to identify which corresponding functional form of the protein may be present or play a role in the secondary assay or model system employed. Such tests can be used to determine the functional form of proteins that may be present in vivo as well as those that may be physiologically relevant. Such modulators may be used to determine which functionally distinct form is involved in a particular phenotypic or physiological function, e.g., a disease.

In certain embodiments, the present invention provides methods for generating in vitro correlations ("IVCs") for physiological properties of in vivo interest. IVC is generated by using in vivo physiological properties of different proteins to establish a spectrum of activity of a compound, e.g., a profile of the effect of a compound on physiological properties of different proteins. The activity spectrum represents an in vivo physiological property and is thus an IVC of a fingerprint of the physiological property.

In certain embodiments, the in vitro correlation is an in vitro correlation with respect to a negative side effect of the drug. In other embodiments, the in vitro correlation is an in vitro correlation for a beneficial effect of a drug.

In certain embodiments, IVC can be used to predict or confirm one or more physiological properties of a compound of interest. The compounds can be tested for their activity against different proteins and the resulting activity profile compared to the activity profile of IVC generated as described herein. The physiological property of the IVC having an activity profile most similar to the activity profile of the compound of interest is predicted and/or confirmed as the physiological property of the compound of interest.

In certain embodiments, IVC is established by assaying the activity of a compound against a different protein or biological pathway, or a combination thereof. Similarly, to predict or confirm the physiological activity of a compound, the activity of the compound may be tested against different proteins or biological pathways, or combinations thereof.

In certain embodiments, the methods of the invention can be used to determine and/or predict and/or confirm the extent to which a compound of interest causes a particular physiological effect. In certain embodiments, the methods of the invention can be used to determine and/or predict and/or confirm the tissue specificity of the physiological effect of a compound of interest.

Cell lines useful in the present invention can be engineered using gene activation or introduction of transgenes (see, e.g., PCT application publication WO/1994/012650). Molecular beacons or fluorescent oligonucleotides can be used to identify cells expressing a protein of interest (see, e.g., U.S. Pat. No.6,692,965 to Shekdar et al, published 2/17/2004 and International application No. PCT/US2005/005080 published as WO/2005/079462). In certain embodiments, the cell or cell line is engineered to express a protein subunit as part of a multimeric protein. In certain more specific embodiments, the cell or cell line is engineered to express at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 subunits of the multimeric protein. In particular embodiments, the activity profile is generated for a plurality of cell lines, wherein each cell line has been engineered to express a different subunit combination of the multimeric protein.

In certain embodiments, the cell or cell line used in the method of producing IVC is a cell or cell line as described herein, e.g., a cell or cell line having substantially uniform physiological properties, a cell or cell line expressing a protein of interest that does not comprise a protein tag, or a cell or cell line having a Z' factor of at least 0.4 in a functional assay or a cell or cell line cultured in the absence of selective pressure, or any combination thereof. In certain embodiments, the different cells or cell lines used are maintained in parallel under substantially the same culture conditions. The robotic methods described herein can be used to maintain and manipulate cells or cell lines to produce IVC. In certain embodiments, the invention provides a cell or cell line for producing IVC, wherein the cell or cell line is cultured in the absence of selective pressure and wherein the expression of at least one protein of the cell does not change by more than 1%, 5%, 10%, 15%, 20%, 25% 30% 35% or 40% within 3 months. In certain embodiments, the expression of at least one protein does not change by more than 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35% or 40% within 4, 5, 6 or more months.

In more specific embodiments, the activity profile of the compound of interest is established by testing the activity of the compound in a plurality of in vitro assays using cell lines engineered to express the multimeric protein, wherein at least two cell lines express different multimeric proteins. In particular embodiments, the different multimeric proteins are different subunit combinations of multimeric proteins. In certain more specific embodiments, the IVC is produced by testing the compound of interest for at least 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, 98%, or at least 99% of the possible subunit combinations of multimeric proteins. In a more specific embodiment, all possible combinations of subunits are tested. In certain embodiments, at least 5, 10, 25, 50, 100, 150, 200, 250, 500, 1000, 2500, 5000, 7500, or at least 10000 subunit combinations are tested.

In certain embodiments, the different multimeric proteins of different cell lines are different forms of the multimeric protein, wherein the different forms differ by a combination of subunits, stoichiometry, splicing, and/or post-translational modifications, including proteolysis.

In certain embodiments, testing a subject panel for multiple functional forms representing a target or related targets for failed and successful drug candidates can be used to correlate a particular target with adverse or undesirable side effects or therapeutic efficacy observed in the clinic. This information can be used to select well-defined targets in HTS or during the development of compounds for drugs with the required minimal off-target activity.

In certain more specific embodiments, the IVC of a compound is generated by testing the compound against different cell lines expressing different combinations of subunits of the multimeric protein. In more specific embodiments, such multimeric proteins include, but are not limited to, receptor protein complexes such as GABA_A、NaV、GABA_BENaC, sweet taste receptor, umami taste receptor and other multimeric proteins described herein.

In certain embodiments, EnaC, GABA are used_AIVC is produced by NaV, sweet taste receptor, umami taste receptor, bitter taste receptor, CFTR or GCC. As described below, IVC of a compound can represent the effect of the compound on a particular physiological parameter. In certain embodiments, the physiological parameter is measured using functional magnetic resonance imaging ("fMRI"). Other imaging methods may also be used. Such other imaging methods include Computed Tomography (CT); a Computed Axial Tomography (CAT) scan; ultrasound optical diffusion imaging (DOI); diffuse Optical Tomography (DOT); an event-dependent optical signal (EROS); near infrared spectroscopy (NIRS); magnetic Resonance Imaging (MRI); magnetoencephalography (MEG); positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT). In certain embodiments, if IVC represents the effect of a compound on the central nervous system ("CNS"), then IVC can be established in the CNS in association with fMRI patterns. IVC can be produced in a variety of tests and models, including human and animal (e.g., livestock and pets) test models, that correlate with the activity of the compounds. Human diseases and disorders are listed, for example, in The Merck Manual, 18 th edition (Hardcover) Mark h.beers (author), Robert s.porter, Thomas v.jones (editor). In the Diagnostic and statistical Manual of Mental Disorders D, for example, authored by the American psychiatric Association (collective authors) Psychosis and disorders as listed in version 4 (revision) of SM-IV-TR.

IVC produced using GABA may be directed against a compound's physiological effects on CNS disorders, anxiety, sedation, Post Traumatic Stress Disorder (PTSD), memory, learning, autism, epilepsy, alcoholism, mood disorders, CNS function, CNS physiology, CNS development and/or CNS aging. The physiological effect may be an enhancement or alleviation of at least one symptom of the disease.

GABA-producing IVC directed to mood, psychological state, feelings (feelings), feeling, or sensation, including: happy, satisfied, euphoric, manic, exciting, anticipating, satisfying, anxious, depressed, angry, afraid, horror, suspicion, love, hate, contradictory mood, apathy, lassitude, guilt, love, sadness, unpleasant, violent, irritable, motivating (drive), motivation, offensive, hostile, irritability, pride, confidence or lack of confidence, lack of confidence, uneasiness, hesitancy; an active, passive, cooperative, uncooperative, helpful, or unassisted attitude; liberty, happiness, achievement, satisfaction (adequacy), non-satisfaction (inadequacy), or restriction; charming, non-charming, sexual, non-sexual, fondness, dislike, lovely, appetizing or unpleasant; a sense of loneliness or a sense of being included/contained; a sense of belonging or not belonging to a social group; feeling of being sleepy, deceived, utilized, misled, helped, encouraged, discouraged; taking suicide feeling into account; risk, criminal, illegal, avoidance response; or collude to kill the sense of guilt.

IVC can also be generated against GABA which provokes a sensory stimulus of positive or negative mood. The positive sensory stimuli may include odor sensations such as sea winds, forests, food, and the like. In certain embodiments, IVCs that can produce IGABA for sedation or sleep, including restful sleep, continuous sleep, deep sleep, light sleep or rapid eye movement sleep (REM sleep), restful sleep, lack of sleep, insomnia, sleep disorders, sleep disturbances or sleepwalking, sleeptalking or dream-eating; memory, learning, interpretation, analysis, thinking, remembering, placing (playing), producing associations, recalling past events, short term memory, long term memory, or cognition; alcohol dependence or addiction or alcoholism; CNS indications, chronic pain, epilepsy, convulsions, addiction, dependence; endocrine/hormone indications, blink conditioning paradigms, lung cancer, prostate cancer, breast and other cancers and malignancies; glucose metabolism reactions, hypoxia, prostaglandin-induced thermogenesis, cardiac baroreceptor reflex and other reflex abnormalities; and psychiatric disorders (including autism).

IVC produced using bitter receptors may be directed to physiological effects on obesity, sugar absorption, glucagon-like polypeptide (GLP) secretion, or blood glucose regulation. The physiological effect may be appetite, overall nutrition, degree or rate of nutrient absorption, obesity (gain or loss), degree or rate of sugar absorption, up-or down-regulation of GLP secretion or blood glucose regulation. Optionally, the IVC may also be associated with bitter taste in food and/or drugs.

In certain embodiments, IVC can be produced using bitter receptors, which are directed to activity in the gastrointestinal tract (e.g., GLP secretion or secretion of other gastrointestinal hormones), glycemic control or balance, diabetes, feeding, hunger, appetite, nutrient absorption, weight loss, energy. In other embodiments, the bitter taste receptor is used to produce IVC that is directed to a desired or undesired bitter/aftertaste of a beverage or food ingredient or drug (including over-the-counter drugs and different stereoisomeric drugs); for food preferences; for eating disorders (e.g., bulimia, anorexia, or diet); for the sensation or taste of the compound; or quality control of taste or bitter taste receptors or GPCR activity of the beverage or food ingredient or drug. In other embodiments, IVC generated using bitter taste receptors may be directed to: neuronal firing or CNS activity in response to an active compound; nausea, vomiting (including nausea caused by drugs or other compounds e.g., chemotherapy-induced nausea and vomiting (CINV)); activity of a compound on a non-bitter GPCR; bitter taste or bitter taste modulating compounds that are active in the oral cavity but inactive elsewhere (e.g., inactive in the gastrointestinal tract) and vice versa. In certain embodiments, compounds that activate, inhibit, or modulate bitter taste receptors in a tissue-specific manner can be used to modulate blood glucose levels, glucose absorption, and/or prevent emesis or CINV, where modulating activity is desired in the gastrointestinal tract but undesirable in the oral cavity. In certain embodiments, bitterness modulating compounds that are active only in the oral cavity may be used in flavor applications where physiological activity in the gastrointestinal tract is undesirable.

The IVC produced using sweet taste receptors may be directed to a physiological effect on appetite, nutrition, nutrient absorption, obesity, sugar absorption, GLP secretion, or blood glucose regulation. The physiological effect may be appetite, overall nutrition, degree or rate of nutrient absorption, obesity (gain or loss), degree or rate of sugar absorption, up-or down-regulation of GLP secretion or blood glucose regulation.

Sweet taste receptors can be used to generate IVC for: delayed/long lasting/aftertaste or aftertaste of sweetening compounds, including natural and artificial high intensity sweeteners (e.g., saccharin, aspartame, cyclamate, mogroside, honey leaf sugar and its ingredients, acesulfame K, neotame (neotame), sucralose, and mixtures thereof); aiming at food preference; for eating disorders (e.g., bulimia, anorexia, or diet); activity in the gastrointestinal tract (e.g., GLP secretion or secretion of other gastrointestinal hormones), glycemic control or balance, diabetes, eating, hunger, appetite, nutrient absorption, weight loss, energy; desired or undesired bitterness/aftertaste of beverages or food or pharmaceutical ingredients (including over-the-counter drugs and drugs in different isomeric forms); the sensation or taste sensation of the compound; neuronal firing or CNS activity in response to an active compound; nausea/vomiting, including nausea caused by drugs or other compounds (e.g., CINV); quality control of taste of beverages, food or pharmaceutical ingredients or GPCR-triggered activity; sweet or sweet taste modulating compounds that are active in the oral cavity but inactive elsewhere (e.g., inactive in the gastrointestinal tract) and vice versa. In certain embodiments, compounds that activate, inhibit, or modulate sweet taste receptors in a tissue-specific manner can be used to modulate blood glucose levels, and/or prevent emesis or CINV, where modulating activity is desired in the gastrointestinal tract but not in the oral cavity. In certain embodiments, bitterness modulating compounds that are active only in the oral cavity may be used in flavor applications where physiological activity in the gastrointestinal tract is undesirable.

If umami receptors are used to produce IVC, the IVC may be directed to physiological effects on appetite, nutrition, nutrient absorption, obesity, sugar absorption, GLP secretion, blood glucose regulation, or amino acid absorption. The physiological effect may be appetite, overall nutrition, degree or rate of nutrient absorption, obesity (acquired or absent), degree or rate of sugar absorption, or up-or down-regulation of GLP secretion or blood glucose regulation.

The umami receptor can be used to generate IVC for: food preference; eating disorders (e.g., bulimia, anorexia, or diet); activity in the gastrointestinal tract (e.g., GLP secretion or secretion of other gastrointestinal hormones), glycemic control or balance, diabetes, eating, hunger, appetite, nutrient absorption, weight loss, or energy; desired or undesired aftertaste of a beverage or food ingredient or a drug (including over-the-counter drugs and different stereoisomeric drugs); the sensation or taste sensation of the compound; neuronal firing or CNS activity in response to an active compound; nausea/vomiting, including nausea caused by drugs or other compounds (e.g., CINV); quality control of taste or GPCR-triggered activity of a beverage or food or pharmaceutical ingredient; umami or umami modulating compounds that are active in the oral cavity but inactive elsewhere (e.g., inactive in the gastrointestinal tract) and vice versa. In certain embodiments, compounds that activate, inhibit, or modulate umami receptors in a tissue-specific manner can be used to modulate blood glucose levels, glucose absorption, and/or prevent emesis or CINV, where modulating activity is desired in the gastrointestinal tract but is undesirable in the oral cavity. In certain embodiments, umami taste modulating compounds that are active only in the oral cavity may be used in flavour applications where physiological activity in the gastrointestinal tract is undesirable.

IVC produced using ENaC may be directed against the physiological effects of the compound on COPD (chronic obstructive pulmonary disease), CF (cystic fibrosis), fertility, IBS (irritable bowel syndrome), crohn's disease, pulmonary edema, or hypertension. The physiological effect may be an exacerbation of the disease or a reduction or amelioration of at least one symptom of the disease. IVC may also represent different salty tastes of the compounds.

The ENaC may also be used to generate IVCs for: food preference; eating disorders (e.g., bulimia, anorexia, or diet); regulation, secretion, quality, clearance, production, viscosity or consistency of mucus; absorption, retention, equilibration, passage or transport of water through epithelial tissues (particularly lung, kidney, vascular tissues, eye, gastrointestinal, small and large intestine); neuronal firing or CNS activity in response to an active compound; pulmonary indication; gastrointestinal indications such as colon cleansing (bowel cleansing), Irritable Bowel Syndrome (IBS), drug-induced (i.e. opioid) constipation, constipation/CIC in bedridden patients, acute infectious diarrhea, escherichia coli, cholera, viral gastroenteritis, rotavirus, regulation of malabsorption syndrome, infantile diarrhea (viral, bacterial, protozoan), HIV or short bowel syndrome; fertility indicators such as sperm motility or sperm capacitation; female reproductive indications, viscous cervical/vaginal secretions (i.e., excessive cervical mucus); contraception, e.g., negatively affecting sperm motility or cervical mucus quality associated with sperm motility; or xerostomia, dry eye, glaucoma or runny nose.

EnaC can also be used to generate IVC for sensory or taste perception of a compound. In particular, IVC may be directed to the taste of salty tastes such as magnesium, sodium, potassium and/or calcium salts. The salts may have different counterions such as sulfate, chloride or other halides, bromide, phosphate, lactate and other salts. In particular embodiments, the salt is potassium chloride or potassium lactate. In certain embodiments, IVC represents taste perception of a combination of different salts.

IVC generated using CFTR is directed against the physiological effects of the compound on COPD (chronic obstructive pulmonary disease), CF (cystic fibrosis), fertility, IBS (irritable bowel syndrome), crohn's disease, pulmonary edema, or hypertension. The physiological effect may be an exacerbation of the disease or a reduction or amelioration of at least one symptom of the disease. IVC may also represent different salty tastes of the compounds.

CFTR can also be used to generate IVC for: food preference; eating disorders (e.g., bulimia, anorexia, or diet); regulation, secretion, quality, clearance, production, viscosity or consistency of mucus; absorption, retention, equilibration, passage or transport of water through epithelial tissues (particularly lung, kidney, vascular tissues, eye, gastrointestinal, small and large intestine); a sensation or taste sensation to the compound; neuronal firing or CNS activity in response to an active compound; pulmonary indication; gastrointestinal indications such as colon cleansing, Irritable Bowel Syndrome (IBS), drug-induced (i.e. opioid) constipation, constipation/CIC in bedridden patients, acute infectious diarrhea, escherichia coli, cholera, viral gastroenteritis, rotavirus, regulation of malabsorption syndrome, infantile diarrhea (viral, bacterial, protozoal), HIV or short bowel syndrome; fertility indicators such as sperm motility or sperm capacitation; female reproductive indications, viscous cervical/vaginal secretions (i.e., excessive cervical mucus); contraception, e.g., negatively affecting sperm motility or cervical mucus quality associated with sperm motility; or xerostomia, dry eye, glaucoma or runny nose; or endocrine indications, i.e., pancreatic function in CF patients.

IVC produced using NaV is directed against physiological effects on pain. The physiological effect may be an exacerbation or reduction of pain.

IVC was generated using NaV for the following: diseases (including chronic pain, acute pain, cardiac pain, muscle pain, bone pain, organ pain, fatigue, pain caused by overstimulation, abrasion, physical injury or internal injury, pain caused by cancer, pain caused by physical injury, sensory pain, phantom pain, and debilitating pain); an action potential, generation or propagation of neuronal signaling, or transmission of neuronal information; or a muscle or cardiac indication or is directed to the in vivo activity of the compound on muscle or cardiac muscle.

The IVC generated using GCC is an IVC for: gastrointestinal indications include: constipation, IBS (irritable bowel syndrome) including IBS-C (constipation), IBS-D (diarrhea) or IBS-M (mixed), chronic idiopathic constipation, opioid or drug-induced constipation, constipation/CIC in bedridden patients, acute infectious diarrhea (e.g., mediated by the bacteria escherichia coli, salmonella (salmonella), cholera, particularly traveler's diarrhea), viral gastroenteritis, clinical indications for rotavirus, modulation of malabsorption syndrome, infantile diarrhea (viral, bacterial, protozoan), short bowel syndrome, colitis (collagenous, lymphocytic), crohn's disease, UC, diverticulitis, cystic fibrosis, or ulcers (including peptic ulcers); regulation/modulation of mucosal and/or epithelial fluid absorption and secretion; lung indications such as cystic fibrosis, renal function, cardiac fibrosis, cardiac hypertrophy, hypertension, eye disorders (i.e. autosomal dominant retinitis pigmentosa and leber congenital amaurosis), growth disorders, short stature, stroke and other vascular injuries; CNS indications such as memory or depression; or inflammatory disorders (i.e., rheumatoid arthritis).

Odorant receptors can be used in IVC to produce sensory stimuli that are directed towards stimulating positive or negative mood. The sensory stimuli that stimulate a positive mood may include a pleasant smell such as sea breeze, forest smell, or food smell. Sensory stimuli that stimulate a negative mood may include unpleasant odors. IVC can be generated against sensory stimuli that stimulate the following positive or negative emotions: happy, satisfied, euphoric, manic, exciting, anticipating, satisfying, anxious, depressed, angry, afraid, horror, suspicion, love, hate, contradictory mood, apathy, lassitude, guilt, love, sadness, unpleasant, violent, irritative, motivation, aggressive, hostile, irritability, pride, confidence or lack of confidence, lack of confidence, uneasy, hesitancy; an active, passive, cooperative, uncooperative, helpful, or unassisted attitude; liberty, happiness, achievement, satisfaction, dissatisfaction, or restriction; charming, non-charming, sexual, non-sexual, fondness, dislike, lovely, appetizing or unpleasant; a sense of loneliness or a sense of being included/contained; a sense of belonging or not belonging to a social group; feeling of being sleepy, deceived, utilized, misled, helped, encouraged, discouraged; taking suicide feeling into account; risk, criminal, illegal, avoidance response; or collude to kill the sense of crime.

Acetylcholine receptors can also be used to produce IVC directed to sensory stimulation that stimulates positive or negative mood. Infection stimuli include, but are not limited to, odors. IVC can be generated against sensory stimuli that stimulate the following positive or negative emotions, mood, mental state, emotion, sensation or perception, including: happy, satisfied, euphoric, manic, exciting, anticipating, satisfying, anxious, depressed, angry, afraid, horror, suspicion, love, hate, contradictory mood, apathy, lassitude, guilt, love, sadness, unpleasant, violent, irritative, motivation, aggressive, hostile, irritability, pride, confidence or lack of confidence, lack of confidence, uneasy, hesitancy; an active, passive, cooperative, uncooperative, helpful, or unassisted attitude; liberty, happiness, achievement, satisfaction, dissatisfaction, or restriction; charming, non-charming, sexual, non-sexual, fondness, dislike, lovely, appetizing or unpleasant; a sense of loneliness or a sense of being included/contained; a sense of belonging or not belonging to a social group; feeling of being sleepy, deceived, utilized, misled, helped, encouraged, discouraged; taking suicide feeling into account; risk, criminal, illegal, avoidance response; or collude to kill the sense of crime.

IVCs that produce acetylcholine receptors for physiological effects of compounds on CNS disorders, anxiety, sedation, PTSD, memory, learning, autism, epilepsy, alcoholism, mood disorders, CNS function, CNS physiology, CNS development and/or aging of the CNS. IVC produced using acetylcholine can also be produced, which is directed to the following: sedentary or sleep, including restful sleep, continuous sleep, deep sleep, light or rapid eye movement sleep, restful sleep, sleeplessness, insomnia, sleep disorders, sleep disturbances or sleepwalking, sleeptalking or dream eating; memory, learning, interpretation, analysis, thinking, remembering, placing, producing associations, recalling past events, short term memory, long term memory, or cognition; alcohol dependence or addiction or alcoholism; CNS indications, chronic pain, epilepsy, convulsions, addiction, dependence; endocrine/hormone indications, blink conditioning paradigms, lung cancer, prostate cancer, breast and other cancers and malignancies; glucose metabolism reactions, hypoxia, prostaglandin-induced thermogenesis, cardiac baroreceptor reflex and other reflex abnormalities; and psychiatric disorders (including autism).

In certain embodiments, the cells or cell lines used in the methods are engineered to express one or more proteins set forth in tables 6 and 7-22 herein.

In certain embodiments, IVC represents a physiological property of a compound in a tissue of an organism, an organ of an organism, an extracellular matrix of an organism, a system of an organism (e.g., an immune system of an organism), or the whole organism. The organism may be a vertebrate. The organism may be a mammal. In a more specific embodiment, the organism is a mouse, rat, dog, cat, cow, horse, donkey, goat, monkey or human. The physiological property may be an effect on a particular cell type, tissue, organ or organ system. For example, the physiological property can be an effect on mammalian tissue, healthy tissue, diseased tissue, cancer tissue, embryonic tissue, adult tissue, transplanted tissue, organ tissue, liver tissue, neuronal tissue, gastrointestinal tissue, muscle tissue, adipose tissue, skin, urogenital tissue, neuronal tissue, central nervous system, cardiovascular tissue, endocrine system, skeletal tissue, bone, immune system, organ, cell, or specialized cell, as well as any other cell disclosed herein. In certain embodiments, the physiological property is tissue protective activity, anti-inflammatory activity, neural stimulation, or has activity similar to that of a substance or compound, including but not limited to: adamantane antiviral agents, adrenergic bronchodilators, agents for hypertensive emergencies, agents for pulmonary hypertension, anti-amebiasis, analgesic compounds (analgesic combination), analgesics, androgens and synthetic steroids, angiotensin II inhibitors, anorectics, antacids, antihelminthics, antiangiogenic ophthalmic formulations, anti-infectives, antianginals, antiarrhythmics, analgesic compounds, antibiotics/antineoplastics, anticholinergic antiemetics, anticholinergic Parkinson's disease agents, anticholinergic bronchodilators, anticholinergic/antispasmodics, anticoagulants, anticonvulsants, antidepressants, antidiabetic agents, antidiarrheal, antidotensive, antiemetics/antidazlocytic agents, antifungal agents, antigout agents, antihyperlipidemic mixtures, anti-pro-drugs, anti-diabetic agents, antidiabetic mixtures, antihyperlipidemic drugs, anti-inflammatory drugs, antihyperlipidemic drugs, anti-emetics, antihypertensive combination, antihyperlipidemic agents, antimalarial agents, quinoline antimalarial agents, antimetabolites, antimigraine agents, antineoplastics, antidotes, antineoplastics interferons, antineoplastics, antiparkinsonism agents, antiplatelet agents, pseudomonadins, antipsoriatic agents, antipsychotic agents, antirheumatic agents, antiseptics and bactericides, antitoxin agents and antidophitoxins, antitubercular agents, antiviral agents, interferon agents, anxiolytic agents, sedatives and hypnotics, bile acid sequestrants, bronchodilators, cardiac stress agents, chelating agents, cholinergic muscle stimulants, central nervous system stimulants, coagulation modifiers (ligands), Contraceptives, decongestants, digestive enzymes, diuretics, dopaminergic antiparkinson agents, drugs used in alcohol dependence, expectorants, factor Xa inhibitors, fatty acid derivative anticonvulsants, functional bowel disorders, gallstone dissolvents, general anesthetics, urogenital tract drugs, GI stimulants, glucose-elevating agents, platelet glycoproteins, growth hormone receptor antagonists, hematopoietic stem cell mobilizers (ematopoietic stem cell mobilizers), heparin antagonists, hormone replacement therapy, hormonal/antineoplastic agents, immunosuppressive agents, impotence agents (impotence agents), in vivo diagnostic biologicals, incretin analogs, inotropic agents, laxatives, anesthetic agents, local anesthetics, pulmonary surfactants, lymphatic stains, lysosomal enzymes, mucolytics, muscle relaxants, mydriatics, glaucoma-eye preparations, pharmaceutical preparations for treating alcohol dependence, drugs for treating liver cancer, liver cancer cell proliferative disorders, liver cancer cell diseases, liver cancer cells, liver, Ophthalmic lubricants and irrigation methods (irrigations), spermicides, vasodilators or vasopressors.

In certain embodiments, IVC represents the physiological effect of a compound of interest on a virus, bacterium, fungus, or yeast. Such compounds are useful, for example, as antibiotics against viruses, bacteria, fungi, or yeast.

Assays that test and/or confirm the activity of a compound of interest against a particular multimeric protein of interest depend on the biological activity of the multimeric protein. Any assay used in the methods of the invention can be performed in a high throughput format.

Exemplary proteins, their references and possible assays are shown in the following table (table 5). Such examples are non-limiting, as the listed assays can be used for targets other than the listed targets, and the listed targets can be tested using other assays besides the listed assays.

TABLE 5

The activity profile of the compound of interest can be compared to the signature activity by calculating a correlation between the activity profiles, such as, but not limited to, calculating a similarity measure between the activity profiles. The labeled activity profile may be one of a set of activity profiles in a database. The signature activity profile may be a historical profile. The database of labeled activity profiles may be stored on a computer readable storage medium. In particular embodiments, the database comprises at least 10 marker activity profiles, at least 50 marker activity profiles, at least 100 marker activity profiles, at least 500 marker activity profiles, at least 1,000 marker activity profiles, at least 10,000 marker activity profiles, or at least 50,000 marker activity profiles, each marker activity profile comprising at least 2, at least 10, at least 100, at least 200, at least 500, at least 1,000, at least 2000, at least 2500, at least 7500, at least 10,000, at least 20,000, at least 25,000, or at least 35,000 component measurands. The activity profile of the compound of interest can include measurements indicative of the effect of the compound of interest on the biological activity of different multimeric proteins, e.g., a first protein subunit and a second protein subunit. The signature activity profile provides an in vitro correlation of known physiological properties of previously characterized compounds. The physiological property may be a pharmacological property such as, but not limited to, a negative side effect of a drug or an effective effect of a drug. Each signature activity profile can include measurements representing the effect of each previously characterized compound on the biological activity of a different multimeric protein, e.g., a first protein subunit and a second protein subunit of a multimeric protein. In certain embodiments, a correlation between the activity profile of the compound of interest and each signature activity profile of a plurality of signature activity profiles stored in a database can be calculated. The correlation can be calculated by comparing a measure in the activity profile of a compound of interest that is indicative of the effect of the compound of interest on the biological activity of a given multimeric protein with a corresponding measure of a signature activity profile that is indicative of the effect of a previously characterized compound on the biological activity of the same multimeric protein. An activity profile of a compound of interest can be considered to correlate with a signature activity profile if the measure in the signature activity profile is within about 2%, about 5%, about 8%, about 10%, about 12%, about 15%, about 20%, about 25%, about 30%, or about 35% of the measure in the activity profile of the compound of interest.

An activity profile of a compound of interest may be considered most similar to a signature activity profile if the measure of similarity between the activity profile of the compound of interest and the signature activity profile is above a predetermined threshold. In particular embodiments, the predetermined threshold may be determined as a value representing a measure of similarity in the signature activity profile measured to within about 2%, about 5%, about 8%, about 10%, about 12%, about 15%, about 20%, about 25%, about 30%, or about 35% of the activity profile of the compound of interest.

p＝[p₁，...p_i，...P_n]

wherein p is_iIs a measure of the ith component, e.g., the effect of the compound of interest on the ith biological activity of a given multimeric protein. In certain embodiments, n is greater than 2, greater than 10, greater than 100, greater than 200, greater than 500, greater than 1000, greater than 2000, greater than 2500, greater than 7500, greater than 10,000, greater than 20,000, greater than 25,000, or greater than 35,000. Each signature activity spectrum can also be represented as a vector p. In calculating the correlation, for each component i 1.. n, the measured quantity of the ith component of the vector representing the activity spectrum of the compound of interest can be compared to the measured quantity of the ith component of the corresponding vector representing the activity spectrum. However, there are many ways in which correlations can be calculated. Indeed, any statistical method known in the art for determining the probability of correlation of two data sets can be used to identify whether a correlation exists between the activity profile of a compound of interest and a signature activity profile according to the methods of the present invention. For example, a similarity measure sim (pi) may be used ₁，pi₂) Calculating the Activity Profile (pi) of the Compound of interest₁) With respective marker Activity Profile (pi)₂) The correlation between them. Computing a similarity measure sim (pi)₁，pi₂) One way of (2) is to calculate the negative 2 power of the euclidean distance. In alternative embodiments, sim (pi1, pi2) may be calculated using measures other than euclidean distance, e.g.Manhattan distance, chebyshev distance, vector angle, correlation distance, normalized euclidean distance, mahalanobis distance, squared pearson correlation coefficient, or minkowski distance. In certain embodiments, the pearson correlation coefficient, squared euclidean distance, squared euclidean sum or squared pearson correlation coefficient is used to determine similarity. Such measures can be calculated, for example, using AS (Statistical Analysis systems institute, Cary, North Carolina) or S-Plus (Statistical Sciences, Inc., Seattle, Washington). The use of such measures is described in Draghici, 2003, Data Analysis Tools for DNA microarray, Chapman&Hall, CRCPress London, chapter 11, which is incorporated herein by reference in its entirety for this purpose.

The correlation may also be calculated based on rank, where x_iAnd y_iIs the ranking of the measured values in ascending or descending numerical order. See, e.g., Conover, practical Nonparametric statics, 2 nd edition, Wiley, (1971). Shannon mutual information may also be used as a similarity measure. See, for example, Pierce, An Introduction To information theory: symbol, Signals, and Noise, Dover, (1980), which is incorporated herein by reference in its entirety.

Various classifiers known in the art can be trained according to the methods described herein and used to classify compounds of interest according to physiological properties (such as, but not limited to, pharmacological properties). Algorithms can be used to generate classifiers that can predict a physiological property of a compound of interest using an activity profile of the compound of interest. Exemplary classifiers are described above. In certain embodiments, the classifier can be trained using the measurands in the signature activity profile of the previously characterized compound and the known physiological properties associated with the previously characterized compound.

The classifier may be an algorithm for classification by using an unsupervised or supervised learning algorithm to estimate the measured and known physiological properties associated with previously characterized compounds in the signature activity profile of the previously characterized compounds. Any standard unsupervised or supervised learning technique known in the art may be used to generate the classifier. The following are non-limiting examples of unsupervised and supervised algorithms known in the art. In view of the disclosure of the present application, one of ordinary skill in the art will appreciate that other pattern classification or regression techniques and algorithms may be used for the classifier and the present invention includes all such techniques.

A neural network. Neural networks (e.g., two-segment regression or classification decision rules) are described above.

And (6) clustering. In certain embodiments, the classifier is learned using clustering. In certain embodiments, a selection component i of the vector representing a signature activity profile is used to cluster the activity profiles. In certain embodiments, prior to clustering, the measurements are normalized to have a mean and unit variance of 0.

Signature activity profiles that show similar measured patterns tend to cluster together throughout the training population. The particular combination of components i that are measured can be considered a good classifier in this aspect of the invention when the vectors are clustered into physiological properties. Clustering is described in Duda 1973 at pages 211-256. As described in section 6.7 of duca 1973, the clustering problem is described as one of the natural groupings found in the data set. To identify natural groupings, 2 problems are to be solved. First, a method of measuring similarity (or dissimilarity) between two activity profiles is determined. This measure (similarity measure) is used to ensure that the activity profiles in one cluster are more similar to each other than they are to other activity profiles. Second, a mechanism for assigning data into clusters using a similarity measure is determined.

Similarity measures are discussed in section 6.7 of Duda 1973, where one method proposed to begin clustering studies is to determine distance functions and calculate a matrix of distances between pairs of activity spectra. If the distance is a good measure of similarity, the distance between the activity spectra in the same cluster is significantly smaller than the distance between the activity spectra in different clusters. However, as mentioned in Duda 1973, page 215, clustering does not require the use of a distance metric. For example, a non-metric similarity function s (x, x ') may be used to compare two vectors x and x'. Conventionally, s (x, x ') is a symmetric function whose value is maximum when x and x' are "similar" in some way. An example of a non-metric similarity function s (x, x') is provided on page 216 of duca 1973.

Other aspects of clustering are discussed further above.

Principal component analysis ("PCA"). In certain embodiments, the classifier is learned using principal component analysis. PCA is discussed above.

In one approach using a PCA learning classifier, vectors representing signature activity spectra can be constructed in the same manner as described above for clustering. In fact, the set of vectors where each vector represents a signature activity spectrum may be considered a matrix. In certain embodiments, the matrix represents a qualitative binary description of monomers in the friedel-wilson method (Kubinyi, 1990, 3D QSAR in drug design methods and applications, pergamonepress, Oxford, pp 589-638, hereby incorporated by reference in its entirety), and is distributed in the space of maximum compression using PCA such that a first Principal Component (PC) may capture the maximum amount of variance information, a second component (PC) captures the second maximum amount of total variance information, and so on until all variance information in the matrix is considered.

Each vector, where each vector represents a member of the training population (e.g., a signature activity profile), is then plotted. Many different types of graphs are possible. In certain embodiments, a one-dimensional map is made. In this one-dimensional plot, the values of the first principal component from each member of the training population are plotted. In this form of the graph, it is expected that an activity profile corresponding to a physiological property will cluster within one range of the first primary component value and a profile corresponding to another physiological property will cluster within a second range of the first primary component value.

Nearest neighbor analysis. Nearest neighbor analysis is described above.

And (5) linear discriminant analysis. In certain embodiments, linear difference analysis is used to learn the classifier. Linear Discriminant Analysis (LDA) attempts to classify subjects into one of two categories based on certain target properties. In other words, the LDA tests whether the target property measured in the experiment predicts the classification of the target. LDA typically requires continuous arguments and discontinuous class dependent variables. In the present invention, the abundance value (abundance value) of a selected combination of vector components i in a subset of the entire training population is used as the necessary continuous argument. The trait subgroup classification (e.g., physiological characteristic) of each member of the training population is used as a discontinuous class dependent variable.

LDA finds a linear combination of variables that maximizes the ratio of inter-group variance to intra-group variance by using grouping information. Clearly, the linear weights used by LDA depend on the degree to which the measures of vector component i are dispersed in the physiological property set throughout the training set. In certain embodiments, LDA is used to train a data matrix of members in a population. The linear discriminants for each member of the training population are then plotted. Ideally, those members of the training population that represent a physiological property would cluster to one range of linear discriminant values (e.g., negative) and those members of the training population that represent another physiological property would cluster to a second range of linear discriminant values (e.g., positive). LDA is considered more successful when the separation between clusters of discrimination values is greater. For more information on linear discriminant analysis, see, e.g., duca, patternressability, 2 nd edition, 2001, John Wiley & Sons, Inc; and Hastie, 2001, The Elements of Statistical Learning, Springer, New York; and Venables & Ripley, 1997, Modern Applied Statistics with s-plus, Springer, New York, each of which is incorporated herein by reference in its entirety.

And (5) performing secondary discriminant analysis. Secondary discriminant analysis is described above.

And a support vector machine. A support vector machine is described above.

And (4) a decision tree. The decision tree is described above.

And (3) a multivariate self-adaptive regression spline method. The multivariate adaptive regression spline method is described above.

Gravity classifier techniques. In one embodiment, a nearest neighbor centroid classifier technique is used. For different physiological properties, such techniques calculate a center of gravity given by the average of the vector components i in the training population (signature activity spectrum) measured, and then assign the vector representing the compound of interest to the class whose center of gravity is closest. This method is similar to k-means clustering except that classes are replaced by known classes. An exemplary implementation of this method is Predictive Analysis of Microarrays (PAM). See, e.g., Tibshirani et al, 2002, Proceedings of the National Academy of Science USA 99; 6567-6572, which are incorporated herein by reference in their entirety.

And (6) regression. In certain embodiments, the classifier is a regression classifier, such as a logistic regression classifier. Such a regression classifier includes coefficients for constructing each activity spectrum of the classifier. In such embodiments, the coefficients of the regression classifier are calculated using, for example, a maximum likelihood method. In such calculations, the measured amount of carrier component i is used.

Other methods of learning the classifier are described further above.

The present invention may be implemented as a computer program product comprising a computer program mechanism embedded in a computer readable storage medium. Furthermore, any of the methods of the present invention may be performed in one or more computers or other forms of devices. Examples of devices include, but are not limited to, computers and measurement equipment (e.g., assay readers or scanners). Furthermore, any of the methods of the present invention can be implemented in one or more computer program products. Some embodiments of the invention provide a computer program product encoding any or all of the methods disclosed in the application. Such methods may be stored on a CD-ROM, DVD, magnetic disk storage product, or any other computer readable or program storage product. Such computer-readable storage media tend to be tangible physical objects (as opposed to carrier waves). Such methods may also be implanted in permanent memory such as ROM, one or more programmable chips, or one or more Application Specific Integrated Circuits (ASICs). Such persistent storage may be located in a server, 802.11 access point, 802.11 wireless bridge/station, repeater, router, mobile phone or other electronic device. Such methods embedded in a computer program product may also be distributed electronically, via the internet, or via transmission of a computer data signal (in which the software module is embedded) either digitally or on a carrier wave (it being clear that such use of the carrier wave is for distribution and not storage).

Some embodiments of the invention provide computer program products including any or all of the program modules shown in fig. 3. These program modules may be stored on a CD-ROM, DVD, magnetic disk storage product, or any other computer readable data or program storage product. Program modules may also be embedded in permanent memory, e.g., ROM, one or more programmable chips, one or more Application Specific Integrated Circuits (ASICs). Such persistent storage may be located in a server, 802.11 access point, 802.11 wireless connection/station, repeater, router, mobile phone or other electronic device. The software modules in the computer program product may also be distributed electronically, via the internet or otherwise, through transmission of a computer data signal (in which the software modules are embedded) either digitally or on a carrier wave.

In particular embodiments, the computer program provides for outputting the results of the claimed method to a user, a user interface device, a computer readable storage medium, a monitor, a local computer, or a computer that is part of a network. Such computer storage media tend to be tangible physical objects (as opposed to carriers).

The invention also provides methods of producing cell lines of well-characterized proteins. For such proteins, there is generally little information about their functional response properties to known compounds. Such lack of established functional benchmarks to assess clonal activity can be a challenge in generating physiologically relevant cell lines. The methods described above provide a way to obtain physiologically relevant cell lines, even for proteins that are not well characterized in the presence of such lack of information. By looking for clones representing many or all functional forms, which may result from the expression of genes comprising proteins, cell lines comprising physiologically relevant forms of proteins can be obtained.

The cells and cell lines of the invention can be used to identify the role of different forms of a protein of interest in different pathological states by correlating the identity of the in vivo form of the protein with the identity of known forms of the protein, based on its response to various modulators. This allows the selection of disease or tissue specific modulators for highly targeted treatment of protein-related pathological conditions.

In which the protein is expected to function under conditions, the cells or cell lines of the invention are contacted with a test compound, and then a statistically significant change in protein activity (e.g., p < 0.05) is detected as compared to a suitable control, e.g., cells not exposed to the test compound. Positive and/or negative controls using known agonists or antagonists and/or cells expressing the protein of interest may also be used. One of ordinary skill in the art will appreciate that various assay parameters, such as signal-to-noise ratio, may be optimized.

In certain embodiments, one or more cells or cell lines of the invention are contacted with a plurality of test compounds, e.g., a library of test compounds. Such libraries of test compounds can be screened using the cell lines of the invention to identify one or more modulators of the protein of interest. The test compound can be a chemical moiety including a small molecule, polypeptide, peptide, peptidomimetic, antibody or antigen-binding portion thereof, natural compound, synthetic compound, extract, lipid, detergent, and the like. In the case of antibodies, they may be non-human antibodies, chimeric antibodies, humanized antibodies or fully human antibodies. The antibody can be a complete antibody, including the complete complement of the heavy and light chains, or an antigen-binding portion of any antibody, including antibody fragments (e.g., Fab and Fab, Fab ', F (ab'), Fd, Fv, dAb, etc.), single chain antibodies (scFv), single domain antibodies, all or an antigen-binding portion of the variable region of the heavy or light chain.

In certain embodiments, the cells or cell lines of the invention may be modified by pretreatment with, for example, enzymes including mammalian or other animal enzymes, plant enzymes, bacterial enzymes, protein modifying enzymes, and lipid modifying enzymes prior to contact with the test compound. Such enzymes may include, for example, kinases, proteases, phosphatases, glycosidases, oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases, bacterial proteases, proteases from the gastrointestinal tract, proteases from the GI tract, saliva, proteases in the oral cavity, proteases from lysed cells/bacteria, and the like. Alternatively, cells and cell lines can be first contacted with a test compound and then enzymatically treated to identify compounds that alter the modification of the protein by the treatment.

In certain embodiments, a large collection of compounds is tested for protein modulating activity in cell-based, functional High Throughput Screening (HTS), for example, using 96-well, 384-well, 1536-well or higher density formats. In certain embodiments, a test compound or a plurality of test compounds, including a library of test compounds, can be screened using more than one cell or cell line of the invention.

In certain embodiments, the cells and cell lines of the invention have increased sensitivity to modulators of the protein of interest. The cells and cell lines of the invention also respond to EC, which has a physiological range for proteins₅₀Or IC₅₀A modifier of value. As used herein, EC₅₀Refers to the induction of a half maximal activation response in a cell or cell lineThe concentration of the compound or substance desired. As used herein, IC₅₀Refers to the concentration of a compound or substance required to induce a half-maximal inhibitory response in a cell or cell line. EC (EC)₅₀And IC₅₀Values can be determined using techniques well known in the art, such as dose response curves relating concentration of a compound or substance to the response of a cell line expressing the protein.

A further advantageous property of the cells and cell lines of the invention is that the modulators identified in the initial screening are functional in a secondary function assay using those cells and cell lines. As will be appreciated by those of ordinary skill in the art, the compounds identified in the initial screening assay must generally be modified, e.g., by combinatorial chemistry, pharmaceutical chemistry, or synthetic chemistry, for their derivatives or analogs to function in a secondary functional assay. However, due to the high physiological relevance of the cells and cell lines of the invention, many of the compounds identified using these cells and cell lines are functional without further modification. In certain embodiments, at least 25%, 30%, 40%, 50% or more of the modulators identified in the initial assay are functional in the secondary assay. In addition, the cell lines of the invention perform in functional assays comparable to the "gold standard" assay. For example, the cell lines of the invention expressing GABA a receptors behave essentially the same in membrane potential assays and electrophysiology.

In other aspects of the invention, differentiated, adult or specialized cells produced according to the invention can be used to produce stem cells. In certain embodiments, cells identified by the methods of the invention, wherein the cell type or specialization is differentiated, adult or specialized cells, can be dedifferentiated into stem cells, including but not limited to pluripotent stem cells, multipotent stem cells, totipotent stem cells, induced totipotent stem cells ("iPS"), embryonic stem cells, cancer stem cells, and organ or tissue specific stem cells. Methods of dedifferentiation are known to those of ordinary skill in the art. See, e.g., Panagiotis a. tsonis; stem Cells from differentiated Cells; molecular interactions 4: 81-83, (2004). Stem cells generated from cells of the invention or cells identified by methods of the invention can be dedifferentiated into a dedifferentiated adult or specialized cell type or specialized cell or cells.

Embryonic stem cells and iPS cells produced from the cells of the invention or identified by the methods of the invention can be used to produce whole non-human organisms such as mice. Methods for producing mice using mouse embryonic stem cells are known to those of ordinary skill in the art. See, e.g., Smith, "EMBRYO-DERIVED STEM CELLS: of Rice and Men ", Annu.Rev.cell Dev.biol.2001, 17: 435-62, which are incorporated by reference herein in their entirety. Methods of using iPS cells to generate mice are known to those of ordinary skill in the art. See, for example, Kang et al, "iPS Cell Can Support Full-development of prefabricated blast-completed emulsions", CellStem Cell, Jul 22, 2009[ electronic pre-press edition ] and ZHao et al, "iPS Cell product visible micro through laminated completion", Nature, July23, 2009[ electronic pre-press edition ].

In certain embodiments, the cells of the invention identified by the methods of the invention in which the cell type or specialization is differentiated, adult or specialized cells can be dedifferentiated into stem cells including, but not limited to, pluripotent stem cells, multipotent stem cells, totipotent stem cells, induced pluripotent stem cells ("iPS"), embryonic stem cells, cancer stem cells, and organ or tissue specific stem cells, and the resulting stem cells can then be differentiated into one or more differentiated, adult or specialized cell types or specific cells.

In certain embodiments, a cell or cell line of the invention, wherein the cell type or specialization is differentiated, adult, or specialized, identified by a method of the invention, can be dedifferentiated into an embryonic stem cell or iPS cell, and the resulting stem cell can then be used to generate an intact organism, such as a mouse. In certain embodiments, cells of the invention identified by methods of the invention in which the cell type or specificity is differentiated, adult, or specialized can be dedifferentiated into embryonic stem cells or iPS cells, and the resulting stem cells can then be used to generate whole organisms such as mice, wherein cells in the same cell type or specificity organism comprise the same properties, such as expression of a protein or RNA of interest, for which the cells of the invention are selected.

In certain embodiments, cells of a specialized cell or tissue type comprising an RNA or protein or a functional or physiological form of an RNA or protein can be used to generate embryonic stem cells or iPS cells useful for generating organisms, such as mice, wherein cells or tissues of the same type of organism comprise the RNA or protein or a functional or physiological form of the RNA or protein. In certain embodiments, the organisms produced comprise the same species of RNA or protein. In other embodiments, the organism produced comprises a different species of RNA or protein. In certain embodiments, the organism is a mouse and the RNA or protein is of human origin. In certain embodiments, the produced organisms comprise the in vitro correlations of the invention. In certain embodiments, the resulting organisms can be used in assays, including preclinical testing. In certain embodiments, the test or preclinical testing is used to predict the activity of a test compound in a human.

In other aspects, the invention provides methods for generating in vitro correlations of physiological properties in vivo. In vitro correlation of physiological properties in vivo includes the effect of one or more compounds on one or more proteins or RNAs expressed in the cells or cell lines of the invention (i.e., expressed in vitro), which correlates with the effect of the one or more compounds on one or more in vivo pharmacological properties. Without being bound by theory, if a test compound is found to have a similar or substantially the same effect on one or more in vitro expressed proteins or RNAs as compared to a reference compound, then the test compound can be considered to have a similar or substantially the same effect on one or more in vivo physiological properties as compared to the reference compound, i.e., the test compound is considered to have a similar or substantially the same in vitro correlation (e.g., at least 90% identical) as compared to the reference compound. In certain embodiments, a test compound is considered to have a similar or substantially the same in vitro correlation (e.g., at least 10%, 20%, 30%, 40%, 50% 60%, 70%, 80%, or 90% identical) as compared to a control compound. In vitro correlations may include one or more activity profiles of the compounds.

In other aspects, the protein or proteins expressed by the cells or cell lines of the invention provide for in vitro correlation of the protein or proteins of interest in vivo. The cells and cell lines of the invention may not be fully reproducible in vivo in providing identical conditions for expression of one or more proteins, e.g., there may be differences in post-translational modification, folding, assembly, subunit combination, transport and/or membrane integration between proteins expressed in the cells or cell lines of the invention as compared to proteins expressed in vivo. Furthermore, parameters used in testing the function of proteins expressed in vitro may not be completely reproducible in vivo, e.g., certain protein function assays performed in vitro may be performed at certain pH or salt concentrations that are considered to be non-physiological. However, the protein expressed by the cells or cell lines of the invention may be biologically active in these non-physiological conditions and may provide at least one functional or pharmacological or physiological property when assayed in vitro. Such a function or pharmacological or physiological property may correspond to a protein in vivo. For example, a compound that modulates or alters a physiological property associated with a protein in vivo may be capable of altering the biological activity of the corresponding protein expressed by the cells or cell lines of the invention when assayed in vitro, thereby establishing a correlation between the protein expressed by the cells or cell lines of the invention and the protein expressed in vivo, and the protein expressed by the cells or cell lines of the invention is considered an "in vivo associate" of the protein expressed in vivo. For example, we have found that different cell lines, each expressing the same set of Nav subunits (α, β 1 and β 2), can respond differently to the same set of compounds. See, for example, example 23 below. These different functional characteristics represent different combinations of subunits in these cell lines, and each of the different combinations of subunits can be considered an in vitro correlation of the corresponding in vivo combination of subunits. Furthermore, we have also obtained a profile of compound activity against a panel of cell lines expressing bitter taste receptors. A test for perception of human taste is used to compare the activity profile of a compound to determine which compound pattern's activity against a group of subjects correlates with a desired taste or aftertaste in vivo.

In addition to response to a compound, the in vitro correlations of the invention can also be generated and/or classified by applying other treatments and/or conditions to the proteins expressed by the cells of the invention. For example, we have generated in vitro correlations of ENaC by proteolysis (e.g., generation of different proteolytic forms of ENaC), and have generated in vitro correlations of sweet/umami receptors by applying different media conditions.

In vitro correlations may include a protein or proteins. Such in vitro correlates may predict the function or activity of their corresponding in vivo proteins. Such in vitro correlations can be used in high throughput screens to identify modulators of one or more biological activities of proteins expressed by the cells or cell lines of the invention (i.e., in vitro correlations), and some or all of the compounds thus identified as in vivo phase substances can also modulate proteins expressed in vivo (e.g., have an in vivo therapeutic effect). In various embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of all compounds identified in the high throughput assay are capable of having a therapeutic effect. In certain embodiments, the in vivo related species may comprise at least 2, 3, 4, 5, or 6 subunits. In certain embodiments, the in vitro correlation may be a heteromultimer. In certain embodiments, the in vitro correlation is stably expressed in cells cultured in the absence of selective pressure. In certain embodiments, the in vitro correlation is expressed in a cell line without causing cytotoxicity. In certain embodiments, the in vitro correlation is expressed in a cell that does not endogenously express the protein or proteins.

In certain embodiments, cells or cell lines expressing in vitro correlations according to the invention can be used to identify modulators of a protein of interest in vivo, e.g., by contacting the cells or cell lines with a test compound; and detecting a change in the activity of the in vitro related protein or proteins in the cell or cell line contacted with the test compound as compared to the activity of the in vitro related protein or proteins in cells not contacted with the test compound, wherein a compound that produces a difference in activity in the presence as compared to the absence is a modulator of the protein of interest in vivo.

In certain aspects, the invention provides kits useful for the methods described herein. In one embodiment, a kit comprises one or more containers containing one or more reagents for use in the methods described herein.

In certain aspects, provided herein are kits useful for the methods described herein. In particular, provided herein are kits comprising one or more cells or cell lines stably expressing one or more complex targets. In certain embodiments, the kits provided herein comprise one or more signaling probes described herein. In particular embodiments, a kit may include one or more vectors encoding one or more complex targets. In particular embodiments, the kit includes one or more dyes for use in functional cell-based assays (e.g., calcium flux assays, membrane potential assays) to screen and select for cells that stably express one or more complex targets.

In certain aspects, provided herein are kits comprising one or more containers comprising one or more reagents and/or cells described herein, e.g., one or more cells, vectors, and/or signaling probes provided herein. In certain embodiments, kits of the invention further comprise control reagents (e.g., control signaling probes, dyes, cells, and/or carriers), wherein such control reagents can be positive control or negative control reagents. Optionally associated with such containers may be a brief introduction to the instructions comprising use of the components of the kit.

Sweet taste receptor and umami taste receptor

The present invention relates to novel cells and cell lines that have been engineered to stably express the T1R2 or T1R3 subunit of the sweet taste receptor, and optionally a G protein. The invention also relates to novel cells and cell lines that have been engineered to stably express the T1R1 and T1R3 subunits of the umami receptor, and optionally the G protein. In certain embodiments, taste receptors (e.g., sweet taste receptors or umami taste receptors) produced in such cells and cell lines are functionally and physiologically relevant. In other aspects, the invention provides methods of making and using such cells and cell lines. The cells and cell lines of the invention comprising a taste receptor, e.g., an umami receptor or a sweet taste receptor, can be used to identify modulators of taste receptors, e.g., umami receptors or sweet taste receptors. Such modulators are useful for altering the taste of, for example, foods or pharmaceuticals, and for the therapeutic treatment of diseases in which taste receptors (e.g., umami or sweet taste receptors) are involved (e.g., obesity and diabetes).

According to some embodiments of the invention, the novel cells and cell lines are transfected individually or doubly with a nucleic acid encoding a sweet taste receptor T1R2 subunit and/or a nucleic acid encoding a sweet taste receptor T1R3 subunit. In some specific embodiments, the novel cells and cell lines are transfected individually or doubly with nucleic acid encoding an umami receptor T1R1 subunit and/or nucleic acid encoding an umami receptor T1R3 subunit. Other subunits may be expressed in the cell from endogenous nucleic acids. The two nucleic acids, if present, may be present in the same or separate vectors. In another embodiment, the novel cells and cell lines of the invention are transfected individually, doubly or triply with nucleic acids encoding the sweet receptor T1R2 subunit, the sweet receptor T1R3 subunit and the G protein. In another embodiment, the novel cells and cell lines of the invention are transfected individually, doubly or triply with nucleic acids encoding the umami receptor T1R1 subunit, the umami receptor T1R3 subunit and the G protein. As before, the nucleic acids may be present in the same or separate vectors. For example, 3 vectors may be used; 1 vector can be used; 2 vectors may be used. Other subunits and optionally G proteins may be expressed in the cell from endogenous nucleic acids. In another embodiment, the novel cells and cell lines activate for expression at least one taste receptor subunit (e.g., an umami receptor subunit or a sweet taste receptor subunit) by gene activation. If desired, additional taste receptor subunits (e.g., umami receptor subunits or sweet taste receptor subunits) and/or optional G proteins can be expressed from the introduced nucleic acid sequence encoding such proteins or can have been expressed from an endogenously active nucleic acid. The novel cell lines of the invention stably express an introduced and/or gene-activated taste receptor subunit (e.g., an umami receptor subunit or a sweet taste receptor subunit) and optionally a G protein.

As described above, in some embodiments of the invention, in addition to producing two subunits of taste receptors, e.g., umami or sweet taste receptors, the cells and cell lines of the invention are engineered to produce G proteins. Cells and cell lines engineered to produce G proteins because they do not produce G proteins in their state prior to being engineered that are necessary to trigger downstream signaling from an activated taste receptor (e.g., an umami receptor or a sweet taste receptor), or the cells are unable to produce sufficient amounts of G proteins for taste receptor-induced signaling, e.g., umami receptor-induced signaling or sweet taste receptor-induced signaling.

In a first aspect, the invention provides cells and cell lines that express taste receptors (e.g., umami or sweet taste receptors) that have enhanced properties compared to cells and cell lines prepared by conventional methods. For example, the taste receptor cells and cell lines of the invention (e.g., umami taste receptor cells and cell lines or sweet taste receptor cells and cell lines) have enhanced expression stability and/or expression levels (even when maintained in culture without selective pressure including, for example, antibiotics and other drugs). In other embodiments, the cells and cell lines of the invention have high Z' values in various assays. In other embodiments, the cells and cell lines of the invention are improved in the context of their expression of physiologically relevant taste receptor activity (e.g., umami receptor activity or sweet taste receptor activity) as compared to more conventionally engineered cells. These properties enhance and improve the ability of the cells and cell lines of the invention to be used in assays to identify modulators of taste receptors (e.g., umami and/or sweet taste receptors) and to improve the functional attributes of the identified modulators.

In various embodiments, the cells or cell lines of the invention express the umami receptor T1R1 and T1R3 subunits or the sweet receptor T1R2 and T1R3 subunits at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 days or more than 200 days at a consistent expression level, wherein consistent expression refers to an expression level that does not change by more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% 9% or 10% over 2 to 4 days of continuous cell culture; (ii) no more than 1%, 2%, 4%, 6%, 8%, 10% or 12% change in continuous cell culture between 5 and 15 days; no more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, or 20% change over 16 to 20 days of continuous cell culture; no more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24% change over 21 to 30 days of continuous cell culture; no more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% change over 30 to 40 days of continuous cell culture; (ii) does not change by more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% over 41 to 45 days of continuous cell culture; no more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% change over 45 to 50 days of continuous cell culture; no more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, or 35% change over 45 to 50 days of continuous cell culture; no more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% or 35% change over a 50 to 55 day continuous cell culture; (ii) does not change by more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35% or 40% over 55 to 75 days of continuous cell culture; (ii) no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% change over 75 to 100 days of continuous cell culture; (ii) does not change by more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% over a 101 to 125 day continuous cell culture; no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% change over a 126 to 150 day continuous cell culture; (ii) no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% change over 151 to 175 days of continuous cell culture; no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% change over 176 to 200 days of continuous cell culture; the change is no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% over more than 200 days of continuous cell culture.

According to the present invention, the taste receptor (e.g., umami receptor or sweet taste receptor) expressed by the cell or cell line of the present invention may be from any mammal, including rat, mouse, rabbit, goat, dog, cow, pig or primate. The T1R2 and T1R3 subunits that together form the expressed sweet taste receptor may be from the same or different species. For example, a sweet taste receptor T1R2 subunit from any species may be co-expressed with a sweet taste receptor T1R3 subunit from the same species or from any other species in a cell or cell line of the invention. Likewise, the T1R1 and T1R3 subunits that together form the expressed umami receptor may be from the same or different species. For example, an umami receptor T1R1 subunit from any species may be co-expressed with an umami receptor T1R3 subunit from the same species or from any other species in a cell or cell line of the invention. Similarly, in embodiments in which the G protein is also expressed in the cells and cell lines of the invention, the G protein may be from any species. Wherein the G proteins are the G proteins mentioned in Table 7. Chimeric G-proteins (Ga15-Ga 16; GNA15-GNA16) may also be expressed in the cells and cell lines of the invention. G proteins from any species may be co-expressed with the sweet taste receptor T1R2 subunit from any species, and any combination of the sweet taste receptor T1R3 subunit from any species or three may be used. In particular embodiments, the sweet taste receptor is a human sweet taste receptor and is preferably characterized by human T1R2 and T1R3 subunits. In another specific embodiment, the umami receptor is a human umami receptor and is preferably characterized by human T1R1 and T1R3 subunits. One aspect of the invention provides a collection of cloned cells and cell lines each expressing the same taste receptor (e.g., umami or sweet taste receptor), or a different taste receptor (e.g., umami or sweet taste receptor). Collections can include, for example, cells or cell lines that express combinations of different subunits or full-length or fragments of such subunits.

The nucleic acid encoding the taste receptor subunits, e.g., the umami receptor subunits T1R1 and T1R2 or the sweet receptor subunits T1R2 and T1R3, and the nucleic acid encoding the optional protein may be genomic DNA, cDNA, synthetic DNA, or mixtures thereof. In certain embodiments, the taste receptor (e.g., umami receptor or sweet taste receptor) subunit-encoding nucleic acid sequence and optionally the nucleic acid sequence encoding the G protein further comprise a tag. Such tags may encode, for example, a HIS tag, a myc tag, a Hemagglutinin (HA) tag, proteins C, VSV-G, FLU, Yellow Fluorescent Protein (YFP), green fluorescent protein, FLAG, BCCP, a maltose binding protein tag, a Nus-tag, Softag-1, Softag-2, a Strep-tag, an S-tag, thioredoxin, GST, V5, TAP, or CBP. The tags can be used to determine taste receptor (e.g., umami receptor or sweet taste receptor) subunit and G protein expression levels, intracellular localization, protein-protein interactions, modulation of taste receptors (e.g., umami receptor or sweet taste receptor), or function of taste receptors (e.g., umami receptor or sweet taste receptor). The tags may also be used to purify or fractionate taste receptors (e.g., umami or sweet taste receptors) or G proteins.

In certain embodiments, the cells or cell lines of the invention may comprise a nucleic acid (SEQ ID NO: 31) encoding the human sweet taste receptor T1R2 subunit. In certain embodiments, the cells or cell lines of the invention may comprise a nucleic acid (SEQ ID NO: 41) encoding the human umami receptor T1R1 subunit. The cells or cell lines of the invention may also comprise a nucleic acid (SEQ ID NO: 32) encoding a human sweet or umami receptor T1R3 subunit. In particular embodiments, the cells or cell lines of the invention comprise nucleic acids encoding T1R3 and T1R1 or T1R 2. In other embodiments, the cells or cell lines of the invention may comprise a nucleic acid sequence (SEQ ID NO: 33) in addition to the nucleic acid encoding the T1R1 and T1R3 subunits or the T1R2 and T1R3 subunits. SEQ ID NO: 33 encodes mouse Ga15 protein. In other embodiments, the G protein is human Ga 15. See SEQ ID NO: 37.

in certain embodiments, the nucleic acid encoding the taste receptor subunit (e.g., umami receptor subunit or sweet taste receptor subunit) and optionally the G protein comprises one or more substitutions, insertions, mutations, or deletions compared to the nucleic acid sequence encoding the wild-type taste receptor subunit (e.g., umami receptor subunit or sweet taste receptor subunit) or G protein. In embodiments that include nucleic acids comprising mutations, the mutations can be random mutations or site-directed mutations. Such nucleic acid changes may or may not result in amino acid substitutions. In certain embodiments, the nucleic acid is a fragment of a nucleic acid that encodes a taste receptor subunit (e.g., an umami receptor subunit or a sweet taste receptor subunit) or a G protein. Nucleic acids encoding polypeptides that retain, as fragments or with such modifications, at least one biological property of a taste receptor subunit (e.g., an umami receptor subunit or a sweet taste receptor subunit) or a G protein, such as its ability to activate the G protein with its other subunits or its ability to be activated by a taste receptor subunit (e.g., an umami receptor subunit or a sweet taste receptor), respectively.

The invention also includes cells and cell lines that stably express a nucleic acid encoding a subunit having a sequence that hybridizes to a sequence selected from the group consisting of SEQ ID NO: 31. SEQ ID NO: 41. SEQ ID NO: 32 or corresponding nucleic acids derived from a species other than human or nucleic acids encoding the same amino acid sequence as any of those nucleic acids have at least about 85% identity. In certain embodiments, the identity of the subunit coding sequence is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more compared to such subunit sequences. The invention also includes cells and cell lines in which a nucleic acid encoding a taste receptor subunit (e.g., an umami receptor subunit or a sweet taste receptor subunit) hybridizes under stringent conditions to a nucleic acid selected from the group consisting of SEQ ID NOs: 31. SEQ ID NO: 41. SEQ ID NO: 32 or corresponding nucleic acids derived from a species other than human or subunit sequences of nucleic acids encoding the same amino acid sequences as any of those nucleic acids.

In certain embodiments, the cell or cell line comprises a taste receptor subunit-encoding nucleic acid sequence (e.g., a nucleic acid sequence encoding an umami receptor subunit or a nucleic acid sequence encoding a sweet taste receptor subunit) that is identical to the nucleic acid sequence of SEQ ID NO: 31. SEQ ID NO: 41. SEQ ID NO: 32 or a corresponding nucleic acid derived from a species other than human or a nucleic acid encoding the same amino acid sequence as any of those nucleic acids comprises at least one substitution. Substitutions may comprise less than 10, 20, 30 or 40 nucleotides or up to or equal to 1%, 5%, 10% or 20% nucleotide sequence. In certain embodiments, the substituted sequence may be identical to SEQ ID NO: 31. SEQ ID NO: 41. SEQ ID NO: 32 or a corresponding nucleic acid derived from a species other than human or a nucleic acid encoding an amino acid sequence identical to any of those nucleic acids (e.g., a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto), or a nucleic acid sequence capable of hybridizing under stringent conditions to SEQ ID NO: 31. SEQ ID NO: 41. SEQ ID NO: 32 or corresponding nucleic acids derived from a species other than human or nucleic acids that encode the same amino acid sequence as any of those nucleic acids.

In certain embodiments, the cell or cell line comprises a nucleic acid sequence encoding a taste receptor subunit (e.g., a nucleic acid sequence encoding an umami receptor subunit or a nucleic acid sequence encoding a sweet taste receptor subunit) comprising the amino acid sequence set forth in SEQ ID NO: 31. SEQ ID NO: 41. SEQ ID NO: 32 or corresponding nucleic acids derived from a species other than human or nucleic acids encoding the same amino acid sequence as any of those nucleic acids. Insertions or deletions may be of less than 10, 20, 30 or 40 nucleotides or up to or equal to 1%, 5%, 10% or 20% of the nucleotide sequence. In certain embodiments, the inserted or deleted sequence can be identical to seq id NO: 31. SEQ ID NO: 41. SEQ ID NO: 32 or a corresponding nucleic acid derived from a species other than human or a nucleic acid encoding an amino acid sequence identical to any of those nucleic acids (e.g., a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto), or a nucleic acid sequence capable of hybridizing under stringent conditions to any of SEQ id nos: 31. SEQ ID NO: 41. SEQ ID NO: 32 or corresponding nucleic acids derived from a species other than human or nucleic acids that encode the same amino acid sequence as any of those nucleic acids.

As noted above, in certain embodiments, the cells and cell lines of the invention optionally express a G protein such as mouse Ga15 protein (SEQ ID NO: 36) or human Ga15 protein (SEQ ID NO: 37). Like the nucleic acid sequences encoding the T1R2 and T1R3 subunits of the sweet taste receptor or the T1R1 and T1R3 subunits of the umami taste receptor, the nucleic acid sequences encoding the G protein (and the amino acid sequence of the G protein) may include substitutions, deletions, and insertions as described above for the sweet taste receptor subunits.

In certain embodiments, the nucleic acid substitution or modification results in an amino acid change, such as an amino acid substitution. For example, SEQ ID NO: 34 (human T1R2), SEQ ID NO: 35 (human T1R3), SEQ ID NO: 42 (umami human T1R1 isoform 1aa), SEQ ID NO: 43 (umami human T1R1 isoform 2aa), SEQ ID NO: 44 (umami human T1R1 isoform 3aa), SEQ ID NO: 45 (umami T1R1 isoform 4aa) or corresponding amino acids derived from a species other than human or SEQ ID NOS: amino acid residues of 36 (mouse Ga15) and 37 (human Ga15) and G proteins from any species may be substituted with conservative or non-conservative substitutions. In certain embodiments, the sequence identity between the original and modified amino acid sequences may differ by about 1%, 5%, 10%, or 20% or differ from the sequence substantially equivalent thereto (e.g., a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto).

A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity) as the parent amino acid residue. In cases where 2 or more amino acid sequences differ from each other by conservative substitutions, the percentage of sequence identity or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Methods for making such adjustments are well known to those skilled in the art. Methods for making this adjustment are well known to those of ordinary skill in the art. See, e.g., Pearson, Methods mol. biol. 243: 307-31(1994).

Conservative modifications in the subunits T1R2 and T1R3 will result in sweet taste receptors having functional and chemical characteristics similar to (i.e., at least 50%, 60%, 70%, 80%, 90%, or 95% identical to) the functional and chemical characteristics of the unmodified sweet taste receptor. Conservative modifications in the subunits T1R1 and T1R3 will result in an umami receptor having functional and chemical characteristics similar to (i.e., at least 50%, 60%, 70%, 80%, 90%, or 95% identical to) the functional and chemical characteristics of the unmodified umami receptor. This also applies to the G protein.

The host cells used to generate the cells or cell lines of the invention may express one or more endogenous taste receptor subunits (e.g., umami or sweet taste receptor subunits) or lack the expression of any taste receptor subunit (e.g., umami or sweet taste receptor subunits) in their native state. This also applies to the G protein. The host cell may be a primary cell, a sperm cell or a stem cell, including an embryonic stem cell. The host cell may also be an immortalized cell. The primary or immortalized host cell may be derived from the mesoderm, ectoderm or endoderm of a eukaryote. The host cell may be an epithelial cell, an epidermal cell, a mesenchymal cell, a neural cell, a renal cell, a hepatic cell, a hematopoietic cell, or an immune cell. For example, the host cell may be a blood/immune cell such as a B cell, a T cell (cytotoxic T lymphocyte, natural killer T cell, regulatory T cell, T helper cell, gd T cell, natural killer cell); granulocytes (basophils, eosinophils, neutrophils/polylobal neutrophils), monocytes/macrophages, erythrocytes (reticulocytes), mast cells, platelets/megakaryocytes, dendritic cells; endocrine cells such as thyroid (thyroid epithelial cells, parafollicular cells), parathyroid (parathyroid chief cells, eosinophils), adrenal (pheochromocyte); cells of the nervous system such as glial cells (astrocytes, microglia), large cell type neuroendocrine cells, astrocytes, nuclear chain cells, burtech cells, pituitary cells (gonadotropic substances, corticotropin-secreting cells, thyrotropin cells, somatotropic cells, prolactin cells), cells of the respiratory system such as lung cells (cells in which type I alveoli are in contact with type II alveoli), clara cells, goblet cells; circulating system cells (cardiomyocytes, pericytes); digestive system cells (stomach (major gastric mucosa cells, parietal cells), goblet cells, panne cells, G cells, D cells, ECL cells, I cells, K cells, enteroendocrine cells, enterochromaffin cells, APUD cells, liver (hepatocytes, kupffer cells), pancreas (β cells, α cells), gall bladder); cartilage/bone/muscle/skin system cells such as osteoblasts, osteocytes, osteoclasts, dental cells (cementoblasts, amelogues); chondrocytes such as chondroblasts, chondrocytes, skin/hair cells such as silk cells, keratinocytes; melanocytes such as muscle cells, adipocytes, fibroblasts; urinary system cells such as podocytes, pericytes, mesangial cells/extrabulbar cells, renal proximal tubule brush border cells, compact plaque cells; reproductive system cells such as sperm, sertoli cells, leydig cells, ova, follicular cells; sensory cells such as cells of the organ of the Corti, olfactory epithelium, temperature-sensitive sensory neurons, Merkel cells, olfactory receptor neurons, pain-sensitive neurons, photoreceptor cells, taste bud cells, hair cells of the vestibular apparatus, carotid body cells. The host cell may be a cell of a eukaryote, a prokaryote, a mammal, a bird, a poultry, a reptile, an amphibian, a frog, a lizard, a snake, a fish, a worm, a squid, a lobster, a sea urchin, a sea cucumber, a sea squirt, a fly, a hydroid, an arthropod, a beetle, a chicken, a lamprey, a rice fish, a zebra finch, a blowfish and a zebra fish. Examples of mammals include humans, non-human primates, bovines, porcines, felines, rats, marsupials, murines, canines, ovines, caprines, rabbits, guinea pigs, and hamsters. The host cell may also be non-mammalian, such as yeast, insect, fungal, plant, eukaryotic, and prokaryotic. Such host cells can provide a more diverse background for testing taste receptor modulators, such as umami receptor modulators or sweet taste receptor modulators (with greater likelihood of the absence of expression products produced by the cell that can interact with the target). In a preferred embodiment, the host cell is a mammalian cell.

Examples of host cells that can be used to generate the cells or cell lines of the invention include, but are not limited to: human embryonic kidney-293T cells, established neuronal cell lines, pheochromocytoma, neuroblastoma, fibroblast, rhabdomyosarcoma, dorsal root ganglion cells, NS0 cells, CV-1(ATCC CCL 70), COS-1(ATCC CRL 1650), COS-7(ATCC CRL1651), CHO-K1(ATCC CCL 61), 3T3(ATCC CCL 92), NIH/3T3(ATCC CCRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1(ATCC CCL 26), MRC-5(ATCC CCL 171), L-cells, HEK-293(ATCC CRL1573) and PC12(ATCC CRL 1721), HEK293T (ATCC CRL-CRL 11268), RBL (ATCC CRL-1378), ATCC-SY 5Y (ATCC CRL-CRL 366), ATCC CCL-3634), SJ-20625 (ATCC CRL-30), HepG2(ATCC HB-8065), ND7/23(ECACC 92090903), CHO (ECACC 85050302), Vero (ATCC CCL 81), Caco-2(ATCC HTB37), K562(ATCC CCL 243), Jurkat (ATCC TIB-152), Per.C6(Crucell, Leiden, The Netherlands), Huvec (ATCC human primary PCS 100. sup. 010, mouse CRL 2514, CRL 2515, CRL 2516), HuH-7D12(ECACC 01042712), 293(ATCC CRL 10852), A549(ATCC CCL 185), IMR-90(ATCC CCL), MCF-7(ATC HTB-22), U-2OS (ATCC HTB-96), T84(ATCC CCL248) or any established cell line (polarized or non-polarized) or can be cultured from a culture Collection such as American type culture Collection (ATCC, Blassas 10801. 2011. sup. 2011. RTM 2200. or European culture Collection (European Collection), salisbury Wiltshire SP40JG England).

In one embodiment, the host cell is an embryonic stem cell, which is then used as the basis for the production of transgenic animals that produce taste receptors, such as umami receptors or sweet taste receptors. Embryonic stem cells that stably express at least one taste receptor subunit (e.g., an umami receptor subunit or a sweet taste receptor subunit) or both receptor subunits (e.g., an umami receptor subunit or two sweet taste receptor subunits) and preferably a functional taste receptor (e.g., an umami receptor or a sweet taste receptor subunit) can be directly implanted into an organism, their nuclei can be transferred to other receptor cells and then implanted into such cells, or they can be used to produce transgenic animals. In certain embodiments, one or more subunits may be expressed in an animal at a desired time and/or tissue-specific expression.

As will be appreciated by one of ordinary skill in the art, any vector suitable for use with the host cell of choice may be used to introduce a nucleic acid encoding a taste receptor subunit (e.g., an umami receptor subunit or a sweet taste receptor subunit) or a G protein into the host cell. The vectors comprising nucleic acids encoding various taste receptor subunits (e.g., umami receptor subunits or sweet taste receptor subunits) or G proteins may be of the same type or may be of different types. Examples of vectors that can be used to introduce nucleic acids encoding taste receptor subunits (e.g., umami receptor subunits or sweet taste receptor subunits) or G proteins into host cells include, but are not limited to, plasmids, viruses, including retroviruses and lentiviruses, cosmids, artificial chromosomes, which can include, for example, pFN11A (BIND) pGL4.31、pFC14A(7)CMVpFC14K(7)CMVpFN24A(7)CMVd3pFN24K(7)CMVd3HaloTag^TMpHT2、pACT、pAdVAntage^TM、pBIND、An enhancer,Promoter, pCI, pCMVTNT^TM、pG5luc、pSI、pTARGET^TM、pTNT^TM、pF12ApF12K RMpReg neo、pYES2/GS、pAd/CMV/V5-DESTVector, pAd/PL-DEST^TMA carrier,pDEST^TM27 carrier, a,pEF-DEST51 vector,pcDNA^TMDEST47 vector, pCMV/Bsd vector, pEF6/His A, B and C, pcDNA^TM2-DEST, pLenti6/TR, pLP-AcGFP1-C, pLPS-AcGFP1-N, pLP-IRE Sneo, pLP-TRE2, pLP-RevTRE, pLP-LNCX, pLP-CMV-HA, pLP-CMV-Myc, pLP-RetroQ, pLP-CMVneo, pCMVScript, pcDNA3.1Hygro, pCDNA3.1neo, pcDNA3.1puro, PSV2neo, pIRUPUR, pSV2 zeo. In certain embodiments, the vector comprises an expression control sequence such as a constitutive or conditional promoter, preferably a constitutive promoter is used. One of ordinary skill in the art would be able to select such sequences. For example, suitable promoters include, but are not limited to, CMV, TK, SV40, and EF-1 α. In certain embodiments, the promoter is inducible, temperature regulated, tissue specific, repressible, heat shock, developmental, cell lineage specific, eukaryotic, prokaryotic, or transient, or a combination or recombination of unmodified mutagenized, randomized, shuffled sequences of any one or more of the above. In other embodiments, the taste receptor subunit (e.g., umami receptor subunit or sweet taste receptor subunit) or more than one of the subunits (and optionally the G protein) is expressed by gene activation or additionally expressed.

In certain embodiments, the vector lacks a selectable marker or drug resistance gene. In other embodiments, the vector optionally comprises a nucleic acid encoding a selectable marker (e.g., a protein conferring drug or antibiotic resistance or more commonly any product that exerts selective pressure on the cell). Each vector used to introduce sequences encoding a different taste receptor subunit (e.g., umami receptor subunit or sweet taste receptor subunit) or G protein has the same or different drug resistance or other selection pressure markers. If more than one drug resistance or the selection pressure markers are the same, simultaneous selection can be achieved by increasing the level of drug. Suitable markers are well known to those of ordinary skill in the art and include, but are not limited to, polypeptide products that confer resistance to any of the following: neomycin/G418, puromycin, hygromycin, bleomycin, methotrexate and blasticidin. Although drug selection (or selection using any other suitable selection marker) is not a necessary step in the generation of the cells and cell lines of the invention, it can be used to enrich the transfected cell population for stably transfected cells, provided that the transfected construct is designed to confer drug resistance. If subsequent selection of cells expressing the umami receptor subunit and optionally the G protein or sweet taste receptor subunit and optionally the G protein is performed using signaling probes, premature selection after transfection may result in some positive cells that may only be transiently transfected rather than stably transfected. However, this effect can be minimized by passaging sufficient cells to allow transient expression in the diluted transfected cells.

In certain embodiments, the vector for introducing a nucleic acid encoding a taste receptor subunit (e.g., an umami receptor subunit or a sweet taste receptor subunit) or optionally a G protein comprises a nucleic acid sequence encoding an RNA tag sequence. An "RNA tag sequence" refers to a nucleic acid sequence that is an expressed RNA or portion of an RNA that can be detected by a signaling probe. The signaling probe can detect multiple RNA sequences. Any of such RNAs may be used as a tag. The signaling probe may be directed to the RNA tag by designing the probe to include a portion complementary to the RNA sequence of the tag. The tag sequence may be a 3' untranslated region of the vector that is co-transcribed and contains the target sequence to which the signaling probe binds. The RNA produced from the nucleic acid of interest may include a tag sequence or the tag sequence may be located in the 5 'untranslated region or the 3' untranslated region. In certain embodiments, the tag is not part of the RNA generated from the nucleic acid of interest. The tag sequence may be present in frame with the protein-encoding portion of the messenger of the nucleic acid of interest or absent therefrom, depending on whether it is desired to tag the protein produced. Thus, for detection by a signaling probe, translation of the tag sequence is not necessary. The tag sequence may comprise multiple target sequences, which may be the same or different, with one signaling probe hybridizing to each target sequence. The tag sequence may encode an RNA having secondary structure. The structure may be a three-arm connection structure. Examples of tag sequences that can be used in the present invention and for which signaling probes can be prepared include, but are not limited to, RNA transcripts of epitope tags such as HIS tags, myc tags, Hemagglutinin (HA) tags, proteins C, VSV-G, FLU, Yellow Fluorescent Protein (YFP), green fluorescent protein, FLAG, BCCP, maltose binding protein tags, Nus-tags, Softag-1, Softag-2, Strep-tags, S-tags, thioredoxin, GST, V5, TAP, or CBP. RNA tag sequences can be readily prepared and used by one of ordinary skill in the art, as described herein.

For the preparation of the cells and cell lines of the invention, it is possible to use, for example, the methods described in U.S. Pat. No. 6,692,965 and in International patent application WO/2005/079462. Both of these documents are incorporated by reference herein in their entirety for all purposes. This technique provides a real-time assessment of millions of cells, such that any desired number of clones (from hundreds to thousands) expressing (i.e., producing RNA) a nucleic acid sequence of interest can be selected. Using cell sorting techniques, such as flow cytometry cell sorting (e.g., with a FACS machine) or magnetic cell sorting (e.g., with a MACS machine), 1 selected cell/well can be automatically deposited in a culture vessel (e.g., a 96-well culture plate) with high statistical reliability. The speed and automation of the technology allows for easy isolation of multigenic recombinant cell lines (i.e., cell lines expressing the T1R2 and T1R3 subunits of taste receptors (e.g., umami or sweet taste receptors) and optionally G proteins).

By using the techniques, the RNA sequence of each taste receptor subunit (e.g., umami receptor subunit or sweet taste receptor subunit) (and optionally a G protein) expressed in a cell or cell line can be detected using signaling probes (also referred to as molecular beacons or fluorescent probes). In certain embodiments, the molecular signal recognizes a target sequence as described above. In another embodiment, the molecular beacon recognizes a sequence within the taste receptor (e.g., umami receptor subunit or sweet taste receptor subunit) (or G protein) itself. The signaling probes can be directed to RNA tags or taste receptor subunit sequences (e.g., umami receptor subunits or sweet taste receptor subunits) (or G protein sequences) by designing the probes to include portions that are complementary to the RNA sequences of the tags or taste receptor subunits (e.g., umami receptor subunits or sweet taste receptor subunits) (or G proteins), respectively.

Nucleic acids encoding taste receptor subunits (e.g., umami receptor subunits or sweet taste receptor subunits) (and optionally nucleic acids encoding G proteins), or sequences encoding taste receptor subunits (e.g., umami receptor subunits or sweet taste receptor subunits) (or G proteins) and tag sequences, and optionally nucleic acids encoding selectable markers, can be introduced into a selected host cell by well-known methods. Gene activation sequences can be introduced into cells in a similar manner using conventional methods well known in the art. Methods include, but are not limited to, transfection, viral delivery, protein or peptide mediated insertion, co-precipitation methods, lipid-based delivery reagents (lipofection), cell transfection agents, lipopolyamine delivery, dendrimer delivery agents, electroporation, or mechanical delivery. Examples of transfection agents are GENEPORTER, GENEPORTER2, LIPOFECTANCE 2000, FUGENENE 6, FUGENE HD, TFX-10, TFX-20, TFX-50, OLIGOFECTAMINE, TRANSFAST, TRANSFECTAM, GENESHUTTLE, TROXENE, GENESILENCER, X-TREMEGENE, PERFECTIN, CYTOFECTIN, SIPORT, UNIFECTOR, SIFECTOR, TRANSIT-LT1, TRANSIT-LT2, TRANSIT-EXPRESS, IFECT, RNHUSTTLE, METAFACTENE, LYOVEC, LIPOTAXI, GENEERASER, GENEJUICE, CYTOPURE, JEI, JETPTPT, MEFEGACTIN, POLYFECTT, TRANSSANGER, RNAFEFEFEFEFEFEFEFEFECTT, FECTEFE, PEI-FETCET, CTIN, and CLONITON METAFECTINE. Taste receptor subunit (e.g., umami receptor subunit or sweet taste receptor subunit) nucleic acid sequences (and potentially nucleic acids encoding G proteins) can be integrated at different locations in a gene of a cell. The expression level of the introduced nucleic acids encoding the taste receptor subunit (e.g., umami receptor subunit or sweet taste receptor subunit) and the G protein can vary based on the site of integration.

After introduction of sequences encoding taste receptor subunits (e.g., umami receptor subunits or sweet taste receptor subunits) or taste receptor (e.g., umami receptor or sweet taste receptor) gene activation sequences into host cells (and optionally introduction of G protein coding sequences into such cells or activation of such sequences) and optionally subsequent drug selection, molecular beacons (e.g., fluorescent probes) are introduced into the cells, and cell sorting is then used to isolate cells that are positive for their signal, and thus for expression of the desired nucleic acid sequence (RNA). It is clear to one of ordinary skill in the art that the sorting can be gated according to any desired expression level. Multiple rounds of sorting can be performed if necessary. In one embodiment, the flow cytometric sorter is a FACS machine. MACS (magnetic cell sorting) or laser ablation of negative cells using laser activated analysis and processing can also be used. According to the present method, cells expressing the sweet taste receptor subunits T1R2 and T1R3 (and optionally the G protein) or the umami receptor subunits T1R1 and T1R3 (and optionally the G protein) are detected and recovered.

Signaling probes useful in the present invention are known in the art and are generally oligonucleotides comprising a sequence complementary to a target sequence and a signal emitting system arranged such that no signal is emitted when the probe is not bound to the target sequence and a signal is emitted when the probe is bound to the target sequence. By way of non-limiting example, a signaling probe may include a fluorophore and a quencher positioned in the probe such that the quencher and fluorophore are brought together in an unbound probe. Upon binding between the probe and target sequence, the quencher and fluorophore separate, resulting in emission of a signal. For example, International publication WO/2005/079462 describes a number of signaling probes that can be used and are preferred for the generation of the cells and cell lines of the present invention. Where a tag sequence is used, the vector for each taste receptor subunit (e.g., umami receptor subunit or sweet taste receptor subunit) (or optional G protein) may comprise the same or different tag sequence. Regardless of whether the tag sequences are identical, the signaling probes may comprise different signaling emitters, e.g., fluorophores with different colors, etc., such that the expression (RNA) of each subunit (and G-protein) can be detected separately. By way of illustration, a signaling probe that specifically detects the sweet taste receptor T1R2mRNA or the umami receptor T1R1mRNA can comprise a red fluorophore, a probe that detects the sweet/umami receptor T1R3 subunit (RNA) can comprise a green fluorophore and, optionally, a probe that detects a G protein (RNA) can comprise a yellow fluorophore. One of ordinary skill in the art will know of other methods for differentially detecting 2 (or optionally 3) expressed RNAs in transfected cells using signaling probes.

Nucleic acids encoding signaling probes can be introduced into a selected host cell by any of a number of methods well known to those of ordinary skill in the art, including, but not limited to, transfection, co-precipitation, lipid-based delivery reagents (lipofection), cytofectins, lipopolyamine delivery, dendrimer delivery, electroporation, or mechanical delivery. Examples of transfection agents are GENEPORTER, GENEPORTER2, LIPOFECTANCE 2000, FUGENENE 6, FUGENE HD, TFX-10, TFX-20, TFX-50, OLIGOFECTAMINE, TRANSFAST, TRANSFECTAM, GENESHUTTLE, TROXENE, GENESILENCER, X-TREMEGENE, PERFECTIN, CYTOFECTIN, SIPORT, UNIFECTOR, SIFECTOR, TRANSIT-LT1, TRANSIT-LT2, TRANSIT-EXPRESS, IFECT, RNHUSTTLE, METAFACTENE, LYOVEC, LIPOTAXI, GENEERASER, GENEJUICE, CYTOPURE, JEI, JETPTPT, MEFEGACTIN, POLYFECTT, TRANSSANGER, RNAFEFEFEFEFEFEFEFEFECTT, FECTEFE, PEI-FETCET, CTIN, and CLONITON METAFECTINE.

In one embodiment, the signaling probe is designed to be complementary to a portion of an RNA encoding a taste receptor subunit (e.g., an umami receptor subunit or a sweet taste receptor subunit) or a portion of a 5 'or 3' untranslated region thereof (or a similar portion of an RNA encoding a G protein). Even if a signaling probe designed to recognize the messenger RNA of interest is capable of detecting falsely endogenously expressed target sequences, the ratio of these is such that the sorter can distinguish between 2 cell types, compared to the ratio of the sequence of interest produced by the transfected cells.

In particular embodiments, signaling probes directed to (e.g., complementary to) the same or other coding exons, non-coding introns, or other sequences within non-coding untranslated sequences may also be designed and used. In particular embodiments, signaling probes directed to components of signaling pathways including sweet taste receptors (e.g., T1R2 and T1R3) or umami taste receptors (e.g., T1R1 and T1R3) may also be used.

The level of expression of a taste receptor subunit (e.g., an umami receptor subunit or a sweet taste receptor subunit) (and optionally a G protein) can vary between cells or cell lines. The expression level of a cell or cell line can also decrease over time due to epigenetic events such as DNA methylation and gene silencing, and loss of copies of the transgene. Such changes can be attributed to a number of factors such as the copy number of the transgene taken up by the cell, the genomic integration site of the transgene, and the integrity of the transgene after genomic integration. Expression levels can be estimated using FACS or other cell sorting methods (i.e., MACS). Additional rounds of introducing signaling probes can be used, for example, to determine whether and to what extent a cell remains positive over time for any one or more of the RNAs against which the cell was initially isolated.

In particular embodiments, cells having different absolute or relative fluorescence levels for at least one signaling probe may be isolated, e.g., by FACS, by gating a subset of cells having appropriate fluorescence levels relative to the entire cell population. For example, the first 5%, first 10%, first 15%, first 20%, first 25%, first 30%, first 35%, first 40%, first 45%, first 50%, first 55%, first 60%, or first 65% of the cells having the highest fluorescence signal for a particular signaling probe (or combination of signaling probes) can be gated and isolated by, e.g., FACS. In other embodiments, the cells with the highest fluorescent signal for a particular signaling probe (or combination of signaling probes) can be gated and isolated, e.g., by FACS, by first 2% to 3%, first 5% to 10%, first 5% to 15%, first 5% to 20%, first 5% to 30%, first 40% to 50%, first 10% to 30%, first 10% to 25%, or first 10% to 50%.

Once cells expressing RNA of T1R1 and T1R3 or T1R2 and T1R3 (and optionally G protein) are isolated, they can be cultured in culture medium under any conditions for a length of time sufficient to produce and identify cells stably expressing subunits (and optionally G protein) (RNA or protein), more preferably expressing functional taste receptors (e.g., umami receptor or sweet taste receptor) (and optionally G protein). In another embodiment of the invention, adherent cells may be adapted to suspension either before or after cell sorting and isolation of individual cells. In one embodiment, the isolated cells can be individually cultured or pooled to produce a population of cells. It is also possible to culture single or multiple cell lines separately or to assemble the cells or cell lines. If the cell or mixture of cell lines stably expresses subunits, more preferably functional taste receptors (e.g., umami or sweet taste receptors), it can be further fractionated until a cell line or group of cell lines having that characteristic is identified. This makes it easier to maintain a large number of cell lines without having to maintain each cell line separately. Thus, positive cells can be enriched from an assemblage of cells or cell lines. At least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the enriched mixed cells are positive for the desired property or activity.

a) providing a plurality of cells expressing mRNA encoding one or more taste receptor subunits, e.g., umami receptor subunits or sweet taste receptor subunits, and optionally a G protein;

b) individually dispersing cells into a single culture vessel, thereby providing a plurality of dispersed cell cultures;

c) culturing the cells under a desired set of culture conditions using an automated cell culture process characterized by substantially identical conditions for each isolated cell culture, normalizing the number of cells in each isolated cell culture during the culturing process, and wherein the isolated cultures are passaged according to the same schedule;

d) determining at least one desired characteristic of a taste receptor (e.g., an umami receptor or a sweet taste receptor) of the isolated cell culture at least 2 times; and

According to this method, the cells are cultured under a desired set of culture conditions. The conditions may be any desired conditions. One of ordinary skill in the art will appreciate which parameters are included within a set of culture conditions. For example, culture conditions include, but are not limited to: culture medium (basal medium (DMEM, MEM, RPMI, serum-free, serum-containing, fully chemically defined, animal-derived free component), monovalent and divalent ion (sodium, potassium, calcium, magnesium) concentrations, additional components added (amino acids, antibiotics, glutamine, glucose or other carbon sources, HEPES, channel blockers, modulators of other targets, vitamins, trace elements, heavy metals, cofactors, growth factors, anti-apoptotic agents), fresh or conditioned medium, with HEPES, pH, specific nutrient depletion or restriction (amino acids, carbon sources)), confluent levels allowed for cells to reach before division/passage ₂Three gas systems (oxygen, nitrogen, carbon dioxide), humidity, temperature, resting or using a shaker, etc., as will be well known to those of ordinary skill in the art.

In certain embodiments, cell confluence is measured and growth rate is calculated from the confluence value. In certain embodiments, cells are dispersed and clots are removed for improved accuracy prior to measuring cell confluence. Methods for making cells monodisperse are well known and can be achieved, for example, by adding a dispersing agent to the culture to be measured. Dispersants are well known and readily available and include, but are not limited to, enzymatic dispersants such as trypsin, or EDTA-based dispersants. Growth rates can be calculated from the confluent date using commercially available software for this purpose, such as HAMILTON VECTOR. Automated confluence measurements, for example using automated microplate readers, are particularly useful. Plate readers for measuring confluence are commercially available and include, but are not limited to, CLONE SELECTIMAGER (Genetix). Typically, at least 2 measurements of cell confluence are made before calculating the growth rate. The number of confluent values used to determine growth rate may be any number convenient or suitable for culture. For example, confluence may be measured multiple times over, for example, 1 week, 2 weeks, 3 weeks, or any time period and at any desired frequency.

When the growth rate is known, according to this method, a plurality of isolated cell cultures are divided into groups by similarity of growth rates. By grouping the cultures into growth rate boxes, the cultures in the groups can be processed together, providing another level of normalization that reduces variation between cultures. For example, cultures in frames may be passaged simultaneously, treated simultaneously with the desired reagents, and so forth. In addition, functional assays generally rely on cell density in the dry assay wells. The true comparison of individual clones was done only by plating them and performing the assay at the same density. Grouping into specific growth rate cohorts enables clones to be plated at a specific density, which allows them to be functionally characterized in a high-throughput format.

The growth rate range in each group may be any convenient range. It is particularly advantageous to select a growth rate range that allows cells to be passaged simultaneously and avoids frequent re-normalization of cell numbers. The growth rate set may include a very narrow range for tight grouping, e.g., average doubling times within 1 hour of each other. But ranges may be as much as 2 hours, as much as 3 hours, as much as 4 hours, as much as 5 hours, or as much as 10 hours, or even broader ranges from each other depending on the method. The need for re-normalization arises when the growth rates in the boxes are not the same, so that the number of cells in some cultures increases faster than others. To maintain substantially equivalent conditions for all cultures in a box, cells must be periodically removed to re-normalize the number across the box. The less the growth rate, the more frequently renormalization is required.

In step d), cells and cell lines may be tested and selected according to any physiological property including, but not limited to: an alteration in a cellular process encoded by the genome; alterations in cellular processes regulated by the genome; a change in a pattern of chromosome activity; a change in a pattern of chromosome silencing; a change in gene silencing pattern; a change in gene activation pattern or efficiency; a change in gene expression pattern or efficiency; alteration of RNA expression pattern or efficiency; changes in RNAi expression pattern or efficiency; alteration of RNA processing pattern or efficiency; alteration in RNA transport pattern or efficiency; a change in protein translation pattern or efficiency; a change in protein folding pattern or efficiency; a change in protein assembly pattern or efficiency; a change in the pattern or efficiency of protein modification; a change in protein transport pattern or efficiency; a change in the mode or efficiency of transport of membrane proteins to the cell surface; a change in growth rate; a change in cell size; a change in cell shape; a change in cell morphology; a change in% RNA content; a change in% protein content; change in% moisture content; changes in% lipid content; a change in ribosome content; a change in mitochondrial content; a change in ER quality; a change in plasma membrane surface area; a change in cell volume; alterations in the lipid composition of the plasma membrane; alteration of lipid composition of the nuclear envelope; changes in the protein composition of the plasma membrane; alteration of the protein composition of the nuclear envelope; a change in the number of secretory vesicles; (ii) a change in the number of lysosomes; a change in the number of cavitation bubbles; alterations in the cells with respect to the following abilities or potentials: protein production, protein secretion, protein folding, protein assembly, protein modification, enzymatic modification of proteins, protein glycosylation, protein phosphorylation, protein dephosphorylation, metabolite biosynthesis, lipid biosynthesis, DNA synthesis, RNA synthesis, protein synthesis, nutrient uptake, cell growth, mitosis, meiosis, cell division, dedifferentiation, conversion into stem cells, conversion into pluripotent cells, conversion into totipotent cells, conversion into stem cell types of any organ (i.e., liver, lung, skin, muscle, pancreas, brain, testis, ovary, blood, immune system, nervous system, bone, cardiovascular system, central nervous system, gastrointestinal tract, stomach, thyroid, tongue, gall bladder, kidney, nose, eye, toenail, hair, taste bud), conversion into any cell type that is differentiated (i.e., muscle, toenail, hair, taste bud), Cardiac muscle, neurons, skin, pancreas, blood, immune, red blood cells, white blood cells, killer T cells, enteroendocrine cells, taste, secretory cells, kidney, epithelial cells, endothelial cells, and also including any of the enumerated animal or human cell types that may be used to introduce nucleic acid sequences), uptake DNA, uptake small molecules, uptake fluorescent probes, uptake RNA, attachment to solid surfaces, adaptation to serum-free conditions, adaptation to serum-free suspension conditions, adaptation to scaled-up cell culture, use in large-scale cell culture, use in drug development, use in high-throughput screening, use in functional cell-based assays, use in membrane potential assays, use in calcium flow assays, use in G protein receptor assays, use in reporter cell-based assays, for use in an ELISA study, for use in an in vitro assay, for use in an in vivo application, for use in a secondary test, for use in a compound test, for use in a binding assay, for use in a panning assay, for use in an antibody panning assay, for use in an imaging assay, for use in a microimaging assay, for use in a multi-well plate, for adaptation to automated cell culture, for adaptation to miniaturized automated cell culture, for adaptation to large scale automated cell culture, for adaptation to cell culture in a multi-well plate (6, 12, 24, 48, 96, 384, 1536 or higher density), for use in a cell chip, for use on a slide, for use on a glass slide, for microarray on a slide or glass slide, for immunofluorescence studies, for use in protein purification, for use in bioproduct production, for use in industrial enzyme production, for use in reagent production for research, for use in vaccine development, for use in cell therapy, for use in transplants or humans, for use in factor isolation secreted by cells, for preparation of cDNA libraries, for purification of RNA, for purification of DNA, for infection by pathogens, viruses or other factors, for resistance to drugs, for suitability maintained under automated miniaturized cell culture bars, for use in protein production for characterization, comprising: protein crystallography, vaccine development, stimulation of the immune system, antibody production, or antibody production or testing. One of ordinary skill in the art will readily recognize suitable tests for any of the above listed characteristics. In particular embodiments, one or more such physical properties can be a consistent physical property associated with a taste receptor (e.g., a sweet taste receptor or an umami taste receptor) and can be used to monitor the expression of a functional taste receptor (e.g., a sweet taste receptor or an umami taste receptor).

Tests that may be used to characterize the cells and cell lines of the invention and/or the matched panel of subjects of the invention include, but are not limited to: amino acid analysis, DNA sequencing, protein sequencing, NMR, testing of protein trafficking, testing of nuclear mass transport, testing of protein subcellular localization, testing of subcellular localization of nucleic acids, microscopy, sub-microscopy, fluorescence microscopy, electron microscopy, confocal microscopy, laser ablation techniques, cell counting, and dialysis. One of ordinary skill in the art understands the method of using any of the above listed tests.

Any automated cell culture system can be used in the methods of the invention. Many automated systems are commercially available and well known to those of ordinary skill in the art. In certain embodiments, the automated system is a robotic system. Preferably, the system includes independently moving channels, a multi-channel head (e.g., 96-point head) and a clamp or preferential pick arm, and a HEPA filtration device to maintain sterility during operation. The number of channels in the pipettor should be suitable for the culture format. A convenient pipette has, for example, 96 or 384 channels. Such systems are known and commercially available. For example, MICROLAB STAR ^TMAn instrument (Hamilton) can be used in the process of the invention. An automated system should be able to perform a variety of desired cell culture tasks. Such tasks will be known to those of ordinary skill in the art. They include, but are not limited to: removing culture medium, replacing culture medium, adding reagents, washing cells, removing wash solution, adding dispersants, removing cells from culture vessels, adding cells to culture vessels, and the like.

The generation of the cells or cell pumps of the invention may comprise any number of isolated cell cultures. However, the advantages provided by this approach increase with increasing cell number. There is no theoretical upper limit on the number of cells or isolated cell cultures that can be utilized in the method. According to the invention, the number of isolated cell cultures may be 2 or more, but more advantageously is at least 3, 4, 5, 6, 7, 8, 9, 10 or more isolated cell cultures, such as at least 12, at least 15, at least 20, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 48, at least 50, at least 75, at least 96, at least 100, at least 200, at least 300 at least 384, at least 400, at least 500, at least 1000, at least 10,000, at least 100,000, at least 500,000 or more.

The cells and cell lines of the invention have enhanced stability in the context of expression and expression levels (RNA or protein) compared to cells and cell lines produced by conventional methods. To identify cells and cell lines with this stable expression profile, the expression of each taste receptor, e.g., umami receptor or sweet taste receptor subunit (and optionally G protein), of the cell or cell line is measured over a time course and the expression levels are then compared. Stable cell lines will continue to express (RNA or protein) the sweet taste receptors T1R2 and T1R3 subunits or the umami taste receptors T1R1 and T1R3 subunits (and optionally the G protein) throughout the time course. In some aspects of the invention, the time course may last for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, etc., or at least 1 month or at least 2, 3, 4, 5, 6, 7, 8, or 9 months or any length of time therebetween.

Isolated cells and cell lines can also be characterized, for example, by qRT-PCR and single-endpoint RT-PCR to determine the absolute and relative amounts of each taste receptor subunit, e.g., umami or sweet taste receptor subunit (or G-protein), that is expressed (RNA). Preferably, the level of amplification of both subunits is substantially the same in the cells and cell lines of the invention.

In other embodiments, the expression of a functional umami receptor or sweet taste receptor (and G protein) is determined over time. In such embodiments, stable expression is measured by comparing the results of the functional assay over time. Assays of cell and cell line stability based on functional assays provide the benefit of identifying cells and cell lines that not only stably express the T1R1 and T1R3 subunits of the umami receptor or the T1R2 and T1R3 subunits of the sweet taste receptor (RNA and optionally the G protein), but also stably produce and appropriately process (e.g., post-translational modifications, subunit assembly, and intracellular localization) the subunits (and the G protein) to produce functional umami receptor or sweet taste receptor.

The cells and cell lines of the invention have the further advantageous property of providing assays with high reproducibility, as evidenced by their Z' factor. See Zhang JH, Chung TD, OldenburgKR, "a Simple statistical parameter for Use in evaluation and Validationof High through screen analysis assays," j.biomol. 4(2): 67-73, which references are incorporated herein by reference in their entirety. The Z' value relates to the quality of the cell or cell line as it reflects the extent to which the cell or cell line will respond consistently to the modulator. Z' is a statistical calculation that takes into account the range of signal-to-noise ratios and signal variability (i.e., from well to well) across the functional response of the multi-well plate to the reference compound. Z' was calculated using data from multiple wells with positive controls and multiple wells with negative controls. The ratio of the standard deviations of their combination is multiplied by 3 to the difference factor and the average is subtracted by 1 to obtain the Z' factor according to the following equation:

If the factor is 1.0, this would indicate an ideal assay with a theoretical maximum of Z', no variability and infinite dynamic range. As used herein, "high Z '" refers to a Z' factor having any fractional number between at least 0.6, at least 0.7, at least 0.75, or at least 0.8, or 0.6 to 1.0. In the case of complex targets such as taste receptors (e.g., umami or sweet taste receptors), high Z 'means a Z' of at least 0.4 or greater. A score close to 0 is undesirable because it indicates that there is overlap between the positive and negative controls. In the industry, for simple cell-based assays, Z ' scores of up to 0.3 are considered marginal scores, Z ' scores between 0.3 and 0.5 are considered acceptable, and Z ' scores above 0.5 are considered excellent. Cell-free or biochemical assays can approach scores for cell-based systems, which tend to be lower because of higher Z 'scores, but Z' cell-based systems are complex.

As will be appreciated by those of ordinary skill in the art, cell-based assays using conventional cells expressing even single-chain proteins typically do not achieve a Z' of greater than 0.5 to 0.6. Even if reported in the art, cells with engineered expression of multi-subunit proteins (from introduced coding sequences or gene activation) would be lower due to their added complexity. Such cells would be unreliable for use in assays, as the results would not be reproducible. On the other hand, the cells and cell lines of the invention have higher Z' values and advantageously produce consistent results in the assay. Indeed, the cells and cell lines of the present invention that express taste receptors (e.g., umami or sweet taste receptors) provide the basis for High Throughput Screening (HTS) compatible assays because they generally have higher Z' values than conventionally produced cells. In certain aspects of the invention, the cells and cell lines result in a Z' of at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, or at least 0.8. For cells expressing taste receptors (e.g., umami or sweet taste receptors), even a Z' value of at least 0.3-0.4 is advantageous because taste receptors (e.g., umami or sweet taste receptors) are polygenic targets. In other aspects of the invention, the cells and cell lines of the invention result in a Z' of at least 0.7, at least 0.75, or at least 0.8, even after the cells have been maintained for multiple passages, e.g., between 5-20 passages, including any integer between 5 and 20. In certain aspects of the invention, the cells and cell lines result in a Z' of at least 0.7, at least 0.75, or at least 0.8 in cells and cell lines maintained for 1, 2, 3, 4, or 5 weeks or 2, 3, 4, 5, 6, 7, 8, or 9 months, including any time period therebetween.

Other advantageous properties of taste receptors (e.g., umami or sweet taste receptors) of the cells and cell lines of the invention are that they stably express the T1R2 and T1R3 subunits of sweet taste receptors or the T1R1 and T1R3 subunits of umami receptors in the absence of drugs or otherwise selective pressure. Thus in a preferred embodiment, the cells and cell lines of the invention are maintained in culture without any selection pressure. In other embodiments, the cells and cell lines are maintained in the absence of any drugs or antibiotics. As used herein, cell maintenance refers to culturing cells after they are selected for their sweet or umami receptor subunit (and optionally G protein) expression as described above. Maintenance does not refer to an optional step of growing the cultured cells under selective pressure (e.g., antibiotics) prior to cell sorting, wherein the introduced marker of the cells allows for enrichment of stable transfectants in the mixed population.

Drug-free and selective pressure-free cell maintenance of the cells and cell lines of the invention provides a number of advantages. For example, drug resistant cells may not express the co-transfected transgene of interest at sufficient levels, as selection depends on the survival of cells that have taken up the drug resistance gene and may or may not contain the transgene. In addition, selection drugs and other selection pressure factors are often mutagenic or otherwise interfere with the physiology of the cell, leading to biased results in cell-based assays. For example, selection of drugs can reduce susceptibility to apoptosis (Robinson et al, Biochemistry, 36 (37): 11169-11178(1997)), increase DNA repair and drug metabolism (Deffie et al, Cancer Res.48 (13): 3595-3602(1988)), increase cell pH (Thiebaut et al, J Histochem Cytomem 38.38 (5): 685 690 (1990); Roepe et al, biochemistry.32 (41): 11042-11056(1993)), Simon et al, Proc Natl Acad Sci USA.91 (3): 1128-1132(1994)), decrease pH of lysosomes and endosomes (Schindler et al, biochemistry.35 (9): 2811-2817 (1996)), Altan et al, J exp.187 (10): 1583): 1998), decrease in plasma membrane potential (11032-11041 (1993)), decrease in cell electrical conductance (ATP 23, 11032 (11041) (Biochem et al, 11041) (11023, 11032-biochem et al), proc NatlAcad Sci usa.90 (1): 312-: 4432-4437(1999)). Thus, the cells and cell lines of the invention allow screening assays that do not have artifacts caused by the selection of drugs. In certain preferred embodiments, the cells and cell lines of the invention are not cultured with a selection pressure factor, such as an antibiotic, either before or after cell sorting, such that cells and cell lines having the desired properties are isolated by sorting even when not starting from an enriched cell population.

In another aspect, the invention provides methods of using the cells and cell lines of the invention. The cells and cell lines of the invention can be used in any application where a functional taste receptor subunit (e.g., an umami receptor subunit or a sweet taste receptor subunit) is desired. Cells and cell lines can be used, for example, in vitro cell-based assays or in vivo assays in which cells are implanted into an animal (e.g., a non-human mammal) to, for example, screen for taste receptor modulators (e.g., umami receptor modulators or sweet taste receptor modulators); producing proteins for crystallography and binding studies; and studying the selectivity and dose of the compounds, receptor/compound binding kinetics and stability, and the effects of receptor expression on cell physiology (e.g., electrophysiology, protein trafficking, protein folding, and protein regulation). The cells and cell lines of the invention can also be used in knock-down studies to examine the effect of a particular taste receptor subunit (e.g., an umami receptor subunit or a sweet taste receptor subunit).

Cells and cell lines expressing various combinations of subunits can be used separately or together to identify taste receptor modulators (e.g., umami receptor modulators or sweet taste receptor modulators). Based on their response to various modulators, cells and cell lines can be used to identify the effects of different forms of taste receptors (e.g., umami or sweet taste receptors) in different taste receptor pathologies by correlating the identity of the in vivo form of the taste receptor with the identity of known forms of taste receptors. This allows for the selection of disease or tissue specific taste receptor modulators (e.g., umami receptor modulators or sweet taste receptor modulators) for highly targeted treatment of such taste receptor-related pathologies.

To identify modulators of taste receptors (e.g., umami receptor modulators or sweet taste receptor modulators), a cell or cell line of the invention can be contacted with a test compound under conditions in which taste receptor (e.g., umami receptor modulator or sweet taste receptor modulator) is expected to function, and then a statistically significant change (e.g., p < 0.05) in taste receptor activity (e.g., umami receptor activity or sweet taste receptor activity) is detected as compared to an appropriate control, e.g., a cell not contacted with the test compound. Positive and/or negative controls employing cells that are known agonists or antagonists and/or express different combinations of taste receptor subunits (e.g., umami receptor subunits or sweet taste receptor subunits) (and optionally G proteins) can also be used. In certain embodiments, the taste receptor activity (e.g., umami receptor activity or sweet taste receptor activity) to be detected and/or measured is calcium release from the endoplasmic reticulum as a result of a downstream signaling event following taste receptor activation (e.g., umami receptor activation or sweet taste receptor activation). One of ordinary skill in the art will appreciate that various assay parameters, such as signal-to-noise ratio, may be optimized.

In certain embodiments, one or more cells or cell lines of the invention can be contacted with a plurality of test compounds, e.g., a library of test compounds. Such libraries of test compounds can be screened using the cell lines of the invention to identify one or more modulators of taste receptors (e.g., umami or sweet taste receptors). The test compound may be a chemical moiety including a small molecule, polypeptide, peptide, peptidomimetic, antibody, or antigen-binding portion thereof. In the case of antibodies, they may be non-human antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies. The antibody can be an intact antibody comprising fully complementary heavy and light chains or an antigen-binding portion of any antibody, including antibody fragments (e.g., Fab and Fab, Fab ', F (ab')₂Fd, Fv, dAb, etc.), single chain antibody (scFv), single domain antibody, all or an antigen binding portion of a heavy or light chain variable region.

In certain embodiments, the cells or cell lines of the invention may be modified by pretreatment with, for example, enzymes including mammalian or other animal enzymes, plant enzymes, bacterial enzymes, protein modifying enzymes, and lipid modifying enzymes prior to contact with the test compound. Such enzymes may include, for example, kinases, proteases, phosphatases, glycosidases, oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases, bacterial proteases, proteases from the gastrointestinal tract, proteases from the GI tract, saliva, proteases in the oral cavity, proteases from lysol cells/bacteria, and the like. Alternatively, cells and cell lines can be first contacted with a test compound and then subjected to enzymatic treatment to identify modified compounds that alter the umami or sweet taste receptor produced by the treatment.

In certain embodiments, a large collection of compounds is tested for taste receptor modulating activity (e.g., umami receptor modulating activity or sweet taste receptor modulating activity) in cell-based, functional High Throughput Screening (HTS), e.g., using 96-well, 384-well, 1536-well or higher density formats. In certain embodiments, a test compound or a plurality of test compounds, including a library of test compounds, can be screened using more than one cell or cell line of the invention. In the case of the cells or cell lines of the invention that express a human sweet taste receptor, the cells or cell lines can be contacted with a test compound to identify compounds that modulate sweet taste receptor activity (either enhanced or reduced) for use in treating a disease or condition characterized by undesired sweet taste receptor activity or reduced desired sweet taste receptor activity or the absence of desired sweet taste receptor activity. Furthermore, according to the methods of the invention, the cells or cell lines of the invention can be used to identify compounds or substances that enhance or inhibit sweet taste for ingestible substances.

In the case of a cell or cell line of the invention that expresses a human umami receptor, the cell or cell line can be contacted with a test compound to identify a modulating compound that modulates (either increases or decreases) umami receptor activity for use in treating a disease or condition characterized by undesired umami receptor activity or reduced or absent desired umami receptor activity. Furthermore, according to the methods of the invention, the cells or cell lines of the invention can be used to identify umami taste enhancing or inhibiting compounds or substances for ingestible substances.

In certain embodiments, the cells and cell lines of the invention have increased sensitivity to modulators of sweet taste receptors. For example, cells and cell lines respond to all known classes of sweetening compounds such as natural sweeteners (e.g., glucose, fructose, and sucrose), high intensity sweeteners (e.g., steviva, rebaudiosides, mogrosides), and artificial sweeteners (e.g., aspartame, saccharin, and acesulfame k)₅₀Or IC₅₀The value activates the G protein.

In certain embodiments, the cells and cell lines of the invention have enhanced sensitivity to modulators of umami receptors. For example, the cells and cell lines of the invention respond to all known classes of umami compounds such as MSG and sodium cyclamate. See, for example, fig. 9-12. The cells and cell lines of the invention also respond to modulators and are in physiological range EC for umami receptors₅₀Or IC₅₀The value activates the G protein.

As used herein, EC₅₀Refers to the concentration of compound or substance required to induce a half-maximal activation response in a cell or cell line. As used herein, an IC ₅₀Refers to the concentration of compound or substance required to induce a half-maximal inhibitory response in a cell or cell line. EC can be determined using techniques well known in the art₅₀And IC₅₀Values, such as dose-response curves relating the concentration of a compound or substance to the response of a cell line expressing an umami receptor or a cell line expressing a sweet taste receptor.

Other advantageous properties of taste receptors (e.g., umami or sweet taste receptors) of the cells and cell lines of the invention are that modulators identified in primary screens using such cells and cell lines are functional in secondary functional assays such as snores or taste tests and sensory evaluations. As will be appreciated by those of ordinary skill in the art, the compounds identified in the initial screening assay must generally be modified, e.g., by combinatorial chemistry, pharmaceutical chemistry, or synthetic chemistry, for their derivatives or analogs to function in a secondary functional assay. However, due to the high physiological relevance of the cells and cell lines of the present invention that express the present taste receptors (e.g., umami or sweet taste receptors), many of the compounds identified using such cells and cell lines are functional without further modification.

In certain aspects, provided herein are methods for efficiently identifying, selecting, and enriching for cells having a desired gene expression profile that confers a desired property (e.g., stable and/or high expression of a functional taste receptor such as a sweet taste receptor, umami taste receptor, or bitter taste receptor) using the high degree of naturally occurring genetic diversity present in the cells. The present methods can identify, select and enrich cells with improved properties from a collection of genetically diverse cells more quickly and efficiently than conventional methods. In particular embodiments, the cell is not genetically modified. In other specific embodiments, the present methods allow for the generation of novel homogeneous populations of cells with improved properties (e.g., more stable and/or higher expression of functional taste receptors such as sweet, umami or bitter taste receptors).

In certain embodiments, the methods described herein comprise selecting a naturally occurring cell having one or more desired properties (e.g., stable and/or high expression of a functional taste receptor, such as a sweet taste receptor, umami taste receptor, or bitter taste receptor). In particular embodiments, the methods described herein comprise selecting cells having naturally occurring variants or mutations in one or more taste receptor genes (e.g., a sweet taste receptor gene or an umami taste receptor gene).

In particular embodiments, the methods described herein comprise selecting a cell that has a naturally-occurring variant or mutation in a promoter region of a gene or a non-coding region of a gene (e.g., an intron, a 5 'untranslated region, and/or a 3' untranslated region). Variants or mutations in the promoter region of a gene or in non-coding regions of a gene may result in higher and/or more stable expression of the gene product. In particular embodiments, variants or mutations in promoter regions of genes or non-coding regions of genes have been modified, for example by methylation or acetylation of DNA. In particular embodiments, the cell comprises an epigenetic modification that affects chromatin remodeling of one or more genes of interest. Non-limiting examples of epigenetic modifications include, but are not limited to, acetylation, methylation, ubiquitination, phosphorylation, and sumoylation.

In certain other embodiments, the method comprises cells undergoing pretreatment. Such pretreatments may be exposed to sunlight or Ultraviolet (UV) light, mutagens such as Ethyl Methane Sulfonate (EMS), and chemical agents. In particular embodiments, such pretreatment may include exposure to undesirable growth conditions, such as hypoxic or low nutrient conditions or toxic conditions.

The methods described herein provide for identifying and/or selecting an isolated cell (e.g., a eukaryotic cell) that expresses one or more genes of interest (e.g., a taste receptor subunit gene, such as a sweet taste receptor subunit gene, an umami taste receptor subunit gene, or a bitter taste receptor subunit gene). In certain embodiments, the gene of interest is expressed at a higher level than other cells as a result of genetic variability. In particular embodiments, the methods described herein comprise (a) introducing into a cell (e.g., a eukaryotic cell) one or more signaling probes capable of detecting an RNA of interest (e.g., capable of hybridizing to a target sequence of an RNA of interest); and (b) determining whether the cell (e.g., eukaryotic cell) comprises the RNA of interest. Such methods may also include quantifying the level of an RNA of interest. In particular embodiments, the methods described herein for identifying a cell having a desired RNA expression profile, wherein the method comprises: (a) introducing into a eukaryotic cell (e.g., a eukaryotic cell) a plurality of signaling probes each capable of detecting an RNA of interest; and (b) quantifying the level of RNA detected by the plurality of signaling probes. The desired gene expression profile can be determined by comparison to a reference population.

In particular embodiments, the methods described herein comprise (a) introducing into a cell (e.g., a eukaryotic cell) one or more signaling probes capable of detecting RNA of a taste receptor (e.g., a sweet taste receptor or an umami taste receptor); and (b) determining whether the cell (e.g., eukaryotic cell) comprises RNA for a taste receptor (e.g., sweet taste receptor or umami taste receptor). Such methods may also include quantifying the level of RNA of a taste receptor (e.g., a sweet taste receptor or an umami taste receptor). In particular embodiments, the methods described herein are used to identify cells having a desired RNA expression profile, wherein the methods comprise: (a) introducing into a eukaryotic cell (e.g., a eukaryotic cell) a plurality of signaling probes each capable of detecting a plurality of RNAs of interest; and (b) quantifying the level of RNA detected by the plurality of signaling probes. The desired gene expression profile can be determined by comparison to a reference population. In particular embodiments, the plurality of RNAs of interest may comprise any combination of the following RNAs: RNA of T1R1, RNA of T1R2, RNA of T1R3, RNA of G protein, and RNA of a gene related to a taste receptor. In certain embodiments, the plurality of RNAs of interest includes RNA of T1R1 and RNA of T1R3 (and optionally RNA of G protein). In certain embodiments, the plurality of RNAs of interest includes RNA of T1R2 and RNA of T1R3 (and optionally RNA of G protein).

In particular embodiments, such methods further comprise the step of comparing the quantified levels of cellular RNA to the levels of RNA in a reference cell, respectively. In particular embodiments, the plurality of signaling probes comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 500, 600, 700, 800, 900, or at least 1000 signaling probes. In certain embodiments, the RNA of interest is translated. In other embodiments, the RNA of interest is not translated. In particular embodiments, the RNA of interest is encoded by a taste receptor subunit gene (e.g., a sweet taste receptor subunit gene, an umami taste receptor subunit gene, or a bitter taste receptor subunit gene). In particular embodiments, the isolated cells (e.g., eukaryotic cells) express one or more recombinant RNAs of interest.

In particular embodiments, eukaryotic cells identified and/or selected by the methods described herein are not genetically engineered (e.g., express one or more transgenes non-recombinantly). In other embodiments, the eukaryotic cells identified and/or selected by the methods described herein have been genetically engineered (e.g., recombinantly express one or more transgenes). In particular embodiments, such cells are somatic cells or differentiated cells.

In other embodiments, the cell comprises a desired gene expression profile. In certain embodiments, the desired gene expression profile is obtained by genetic engineering or by increasing genetic variability. In particular embodiments, the desired gene expression profile can be determined based on a comparison to the gene expression profile of a reference population.

In certain embodiments, the methods described herein are used to identify and/or select cells that express an RNA of interest at a higher level than the average heterogeneous cell population. In certain embodiments, the methods described herein are used to identify and/or select cells that express an RNA of interest (e.g., an RNA of a sweet taste receptor or an umami taste receptor) at a higher level than the average heterogeneous cell population (e.g., an unsorted cell line, e.g., an unsorted population of 293T cell lines). In certain embodiments, the methods described herein are used to identify and/or select cells that express an RNA of interest (e.g., an RNA of a sweet or umami receptor) at a level that is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% higher than the average heterogeneous cell population (e.g., an unsorted cell line, e.g., an unsorted population of 293T cell lines). In certain embodiments, the methods described herein are used to identify and/or select cells that express an RNA of interest (e.g., an RNA of a sweet or umami receptor) at a level that is at least 1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher than the average heterogeneous cell population (e.g., an unsorted cell line, e.g., an unsorted population of 293T cell lines).

In particular embodiments, the methods described herein are used to identify and/or select cells that express an RNA of interest at a lower level than the average heterogeneous cell population. Exemplary heterogeneous cell populations can be a cell population of a mixed cell type of different origin, a cell population of a cell of one cell type that is genetically heterogeneous, or a cell population of one particular cell line from which isolated cells are obtained using the methods described herein.

In particular embodiments, the cells isolated using the methods described herein are cell clones from a cell line. In certain embodiments, the cells isolated using the methods described herein are primary cells. In certain embodiments, the cell isolated using the methods described herein is a transformed cell.

In certain embodiments, the cell is not a human cell. In particular embodiments, the cell is a cell derived from a mouse, rat, monkey, dog, cat, pig, sheep, goat, horse, chicken, frog, worm, insect (e.g., fly), fish, shellfish, or cow. In certain embodiments, the cell is a mammalian cell or a eukaryotic cell. In other embodiments, the cell is a human cell. In certain embodiments, the cell is a primary cell. In other embodiments, the cell is a cell clone of a transformed cell or cell line.

In a particular aspect, provided herein is a method for isolating a cell endogenously expressing a sweet taste receptor T1R2 subunit and/or a sweet taste receptor T1R3 subunit, wherein the method comprises the steps of:

a) providing a population of cells;

b) introducing into a cell a signaling probe or molecular beacon that detects expression of T1R2 and/or introducing into a cell a molecular beacon that detects expression of T1R 3; and

c) isolating cells expressing the sweet taste receptor T1R2 subunit and/or the sweet taste receptor T1R3 subunit.

In another specific aspect, provided herein is a method for isolating cells endogenously expressing a sweet taste receptor T1R2 subunit and a sweet taste receptor T1R3 subunit, wherein the method comprises the steps of:

a) providing a population of cells;

b) introducing a molecular beacon which detects expression of T1R2 into a cell and/or introducing a molecular beacon which detects expression of T1R3 into a cell; and

In particular embodiments of such methods, the population of known cells does not express T1R2 or T1R 3. In other specific embodiments of such methods, any expression level of T1R2 or T1R3 in the isolated cell is at least 10-fold, 50-fold, 100-fold, 250-fold, 500-fold, 750-fold, 1000-fold, 2500-fold, 5000-fold, 7500-fold, 10000-fold, 50000-fold, or at least 100000-fold in the average cell of the population of cells. The level of expression of taste receptor subunits such as T1R2 or T1R3 can be readily determined using methods known to those of ordinary skill in the art.

In a specific aspect, provided herein is a method for isolating a cell endogenously expressing an umami receptor T1R1 subunit and/or an umami receptor T1R3 subunit, wherein the method comprises the steps of:

a) providing a population of cells;

b) introducing into a cell a signaling probe or molecular beacon that detects expression of T1R1 and/or introducing into a cell a molecular beacon that detects expression of T1R 3; and

c) isolating cells expressing the umami receptor T1R1 subunit and/or the umami receptor T1R3 subunit.

In another specific aspect, provided herein is a method for isolating cells endogenously expressing the umami receptor T1R1 subunit and the umami receptor T1R3 subunit, wherein the method comprises the steps of:

a) providing a population of cells;

b) introducing a molecular beacon which detects expression of T1R1 into a cell and/or introducing a molecular beacon which detects expression of T1R3 into a cell; and

In particular embodiments of such methods, the population of known cells does not express T1R1 or T1R 3. In other specific embodiments of such methods, any expression level of T1R1 or T1R3 in the isolated cell is at least 10-fold, 50-fold, 100-fold, 250-fold, 500-fold, 750-fold, 1000-fold, 2500-fold, 5000-fold, 7500-fold, 10000-fold, 50000-fold, or at least 100000-fold in the average cell of the population of cells. The level of expression of taste receptor subunits such as T1R1 or T1R3 can be readily determined using methods known to those of ordinary skill in the art.

Non-limiting examples of methods for determining the expression level of a protein include immunoblotting (Western blotting), flow cytometry, or ELISA. Non-limiting examples of methods for determining RNA expression levels include Northern blotting, RT-PCR, and real-time quantitative PCR. Such protein or RNA expression levels can be determined for a typical population of cells to determine the average expression level of the population.

In other specific embodiments of such methods, the genetic variability of the cell population has been increased prior to the isolating step. In particular embodiments, provided herein are isolated cells produced according to such methods.

Bitter taste receptors

The present application relates to novel cells and cell lines that have been engineered to express one or more bitter taste receptors. In certain embodiments, the novel cells or cell lines of the invention express one or more functional bitter taste receptors. In other aspects, the invention provides methods of making and using the novel cells and cell lines.

According to one embodiment of the invention, the novel cells and cell lines are transfected with nucleic acids encoding natural bitter taste receptors. In other embodiments, the novel cells and cell lines are transfected with nucleic acids encoding allelic variants (i.e., polymorphisms) or mutated bitter taste receptors of the natural bitter taste receptor. In another embodiment, the novel cells and cell lines have been activated for expression by gene activation such that the bitter taste receptor is activated.

In particular embodiments, the novel cells and cell lines express endogenous bitter taste receptors as a result of activation of engineered genes (i.e., activation of expression of endogenous genes), wherein the activation does not occur naturally in cells without appropriate treatment. Engineered gene activation can turn on expression of endogenous bitter taste receptors, for example, in cases where the endogenous bitter taste receptors are not expressed in cell lines without appropriate treatment. Alternatively, the engineered gene activation may result in increased expression levels of endogenous bitter taste receptors, e.g., where the expression levels of endogenous genes in the cell line are undesirably low in the absence of appropriate treatment, e.g., insufficient for functional assays of bitter taste receptors in the cell line. Alternatively, engineered gene activation can be used to overexpress endogenous bitter taste receptors, e.g., for isolating endogenous bitter taste receptors from cell lines. Engineered gene activation can be achieved by a number of methods known to those skilled in the art. For example, one or more transcription factors or transactivators that overexpress or induce transcription of an expressed gene can be obtained by introducing into a cell a nucleic acid that expresses the transcription factors or transactivators under the control of a constitutive or inducible promoter. If the endogenous gene is known to be under the control of an inducible promoter, expression can be induced by contacting the cell with a known inducer of the gene. In addition, nucleic acids encoding the endogenous gene itself can be introduced into the cell to achieve increased levels of gene expression (due to increased copy number in the genome). In addition, the expression of an endogenous gene can be increased using techniques well known in the art, such as RNAi to knock down or even knock down certain known inhibitors of the expression of the endogenous gene by the cell.

Cells and cell lines of the invention comprising a bitter taste receptor, a mutant form thereof, or a naturally occurring allelic variant thereof can be used to identify modulators of bitter taste receptor function, including modulators specific for a mutant form or naturally occurring allelic variant of a particular bitter taste receptor. Cells and cell lines can thus be used to obtain information about the nature, activity and effect of a single natural or mutated form or naturally occurring allelic variant of a bitter taste receptor, and to identify bitter taste receptor modulators that have activity for a particular natural or mutated form or naturally occurring allelic variant or for a subset of natural or mutated forms or naturally occurring allelic variants. Such modulators are useful as therapeutic agents targeting differentially modified bitter taste receptor forms in disease states or tissues. Because polymorphisms of bitter taste receptors in vivo may, for example, contribute to undesirable activity or disease states, the cells and cell lines of the invention may also be used to screen for modulators for therapeutic use where alteration of the response of a mutant form or naturally occurring allelic variant may be desired. The cells and cell lines are also useful for identifying modulators that are active only on natural or mutated forms of bitter taste receptors or subgroups of naturally occurring allelic variants.

The present invention also identifies and addresses difficulties in generating cells and cell lines that express stable bitter taste receptors. As disclosed herein, we have found that expression of a labeled bitter receptor results in incorrect partitioning of the bitter receptor agonist. Specific tags, signal sequences and/or chaperones have been suggested to be essential for the expression and/or transport of bitter taste receptors to the cell surface (Reichling, Meyerhof and Behrens, J.Neurochem.106: 1138-. This creates a dilemma and can lead to the identification of physiologically unrelated modulators of labeled receptors. Thus, the physiologically relevant cell lines of the invention can also facilitate identification of ligands for orphan bitter taste receptors and analysis of the specificity of different receptor-ligand interactions.

In a first aspect, the invention provides cells and cell lines that stably express one or more bitter taste receptors. In certain embodiments, the expressed bitter taste receptor increases intracellular free calcium when activated by an agonist. In certain embodiments, the enhancer, agonist, or activator may be a small molecule, chemical moiety, polypeptide, antibody, or food extract. In other embodiments, the expressed bitter taste receptor reduces intracellular free calcium when inhibited by the antagonist. In certain embodiments, the inhibitor, antagonist, or blocker may be a small molecule, chemical moiety, polypeptide, antibody, or food extract. An enhancer, agonist, activator, inhibitor, antagonist or blocker may act on all or a particular subset of bitter taste receptors. In other embodiments, the cells and cell lines of the invention that express bitter taste receptors have enhanced properties compared to cells and cell lines prepared by conventional methods. For example, cells and cell lines expressing bitter taste receptors have enhanced expression stability (even when cultured in the absence of selective antibiotics) and result in high Z' values. In other aspects, the invention provides methods of making and using cells and cell lines that express bitter taste receptors.

In various embodiments, the cells or cell lines of the invention express bitter taste receptors at a consistent expression level for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 days or more than 200 days, wherein consistent expression refers to an expression level that:

no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% change over 2 to 4 days of continuous cell culture; no more than 2%, 4%, 6%, 8%, 10% or 12% change over 5 to 15 days of continuous cell culture; no more than 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, or 20% change over 16 to 20 days of continuous cell culture; no more than 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24% change over 21 to 30 days of continuous cell culture; no more than a 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% change over 30 to 40 days of continuous cell culture; no more than a 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% change over 41 to 45 days of continuous cell culture; no more than a 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% change over 45 to 50 days of continuous cell culture; no more than 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30% or 35% change over 45 to 50 days of continuous cell culture; no more than a 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% change over 50 to 55 days of continuous cell culture; no more than 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30% or 35% change over 50 to 55 days of continuous cell culture; no more than a 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 35%, or 40% change over 55 to 75 days of continuous cell culture; (ii) no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% change over 75 to 100 days of continuous cell culture; (ii) does not change by more than 1%, 2%, 3%, 4%, 5%, 6%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% over 101 to 125 days of continuous cell culture; no more than a 1%, 2%, 3%, 4%, 5%, 6%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% change over a 126 to 150 day continuous cell culture; no more than 1%, 2%, 3%, 4%, 5%, 6%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% change over 151 to 175 days of continuous cell culture; no more than 1%, 2%, 3%, 4%, 5%, 6%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% change over 176 to 200 days of continuous cell culture; the change is no more than 1%, 2%, 3%, 4%, 5%, 6%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% over more than 200 days of continuous cell culture.

According to the invention, the bitter taste receptor expressed by the cell or cell line may be from any mammal, including rat, mouse, rabbit, goat, dog, cow, pig or primate. In a preferred embodiment, the bitter taste receptor is a human bitter taste receptor.

In certain embodiments, the cells or cell lines of the invention may comprise: a nucleotide sequence encoding human TAS2R1 (SEQ ID NO: 51); a nucleotide sequence encoding human TAS2R3 (SEQ ID NO: 52); a nucleotide sequence encoding human TAS2R4 (SEQ ID NO: 53); a nucleotide sequence encoding human TAS2R5 (SEQ ID NO: 54); a nucleotide sequence encoding human TAS2R7 (SEQ ID NO: 55); a nucleotide sequence encoding human TAS2R8 (SEQ ID NO: 56); a nucleotide sequence encoding human TAS2R9 (SEQ ID NO: 57); the nucleotide sequence encoding human TAS2R10 (SEQ ID NO: 58), the nucleotide sequence encoding human TAS2R13 (SEQ ID NO: 59), the nucleotide sequence encoding human TAS2R14 (SEQ ID NO: 60), the nucleotide sequence encoding human TAS2R16 (SEQ ID NO: 61), the nucleotide sequence encoding human TAS2R38 (SEQ ID NO: 62), the nucleotide sequence encoding human TAS2R39 (SEQ ID NO: 63), the nucleotide sequence encoding human TAS2R40 (SEQ ID NO: 64), the nucleotide sequence encoding human TAS2R41 (SEQ ID NO: 65), the nucleotide sequence encoding human TAS2R43 (SEQ ID NO: 66), the nucleotide sequence encoding human TAS2R44 (SEQ ID NO: 67), the nucleotide sequence encoding human TAS2R45 (SEQ ID NO: 68), the nucleotide sequence encoding human TAS2R46 (SEQ ID NO: 69), the nucleotide sequence encoding human TAS2R47 (SEQ ID NO: 70), A nucleotide sequence (SEQ ID NO: 71) encoding human TAS2R48, a nucleotide sequence (SEQ ID NO: 72) encoding human TAS2R49, a nucleotide sequence (SEQ ID NO: 73) encoding human TAS2R50, a nucleotide sequence (SEQ ID NO: 74) encoding human TAS2R 55; a nucleotide sequence encoding human TAS2R60 (SEQ ID NO: 75); or any combination thereof.

In certain embodiments, the cells or cell lines of the invention may comprise the following polynucleotide sequences: human TAS2R1(SEQ ID NO: 77); human TAS2R3(SEQ ID NO: 78); human TAS2R4(SEQ ID NO: 79); human TAS2R5(SEQ ID NO: 80); human TAS2R7(SEQ ID NO: 81); human TAS2R8(SEQ ID NO: 82); human TAS2R9(SEQ ID NO: 83); human TAS2R10(SEQ ID NO: 84); human TAS2R13(SEQ ID NO: 85); human TAS2R14(SEQ ID NO: 86); human TAS2R16(SEQ ID NO: 87); human TAS2R38(SEQ ID NO: 88); human TAS2R39(SEQ ID NO: 89); human TAS2R40(SEQ ID NO: 90); human TAS2R41(SEQ ID NO: 91); human TAS2R43(SEQ ID NO: 92); human TAS2R44(SEQ ID NO: 93); human TAS2R45(SEQ ID NO: 94); human TAS2R46(SEQ ID NO: 95); human TAS2R47(SEQ ID NO: 96); human TAS2R48(SEQ ID NO: 97); human TAS2R49(SEQ ID NO: 98); human TAS2R50(SEQ ID NO: 99); human TAS2R55(SEQ ID NO: 100); human TAS2R60(SEQ ID NO: 101); or any combination thereof.

The nucleic acid encoding a bitter taste receptor may be genomic DNA or cDNA. In certain embodiments, the nucleic acid comprises one or more mutations that may or may not result in an amino acid substitution compared to a nucleic acid sequence encoding a wild-type bitter taste receptor. In some other embodiments, the nucleic acid comprises one or more naturally occurring allelic variants as compared to the nucleic acid sequence encoding a particular bitter taste receptor that is most frequently present in a given population. Naturally occurring allelic variants include different amino acid sequences of the same bitter taste receptor that occur naturally, e.g., different amino acid sequences of the same bitter taste receptor that are observed in a given population as a result of allelic variation or polymorphism.

Polymorphisms are a common phenomenon in human genes. Polymorphisms present in or near bitter taste receptor genes can alter their function by affecting their expression or can be by, for example, up-or down-regulating their expression level or by altering their amino acid sequence. Table 20 shows reference numbers for unique polymorphisms, including single nucleotide polymorphisms ("SNPs") associated with the human TAS2R gene, the location of SNPs in each reference sequence, and a description of the SNPs. The reference numbers may be retrieved in the Single nucleotide polymorphism database ("dbSNP") of the national center for Biotechnology information ("NCBI"; Bethesda, Md.).

Allelic variations of the human bitter taste receptor gene that result in diversity of the coding sequence have been studied and documented. See, for example, Ueda et al, "Identification of coding strand polynucleotides in human taste receiver genes inventingbitter stating", Biochem Biophys Res Commun 285: 147-; wooding et al, "Natural selection and molecular evolution in PTC, abitter-taste receiver gene", am.J.hum, Genet.74: 637-646, 2004; and Kim et al, "world laptop diversity and coding sequence variant at Human bitmap receiver location", Human Mutation 26: 199, 204, 2005. Table 21 is a list of natural variations in the coding sequences of different human bitter taste receptors. The human bitter taste receptors, their SEQ ID NOS coding sequences and the protein sequence are listed in column 3 above. Nucleotide changes and their positions within each coding sequence identified by their SEQ ID NOS are shown in the columns under "nucleotide changes" and "positions of nucleotide changes", respectively. Amino acid changes within each bitter receptor as identified by their SEQ ID NOS are shown in the column under "description" with one letter abbreviations. For each corresponding SEQ ID NO, their positions are indicated in the column below the "position of amino acid change". In addition, the "description" column also includes identifiers of variants that can be retrieved at the NCBI's dbSNP. "feature identifier" is a unique and stable feature identifier assigned to some variant by the UniProt Protein Knowledgebase, hosted by the European bioinformatics institute (Cambridge, United Kingdom). They can be retrieved in UniProt. "NA" denotes an identifier that has not been assigned by UniProt.

Changes in human taste are well known phenomena. Without wishing to be bound by theory, changes in bitter taste may be associated with polymorphisms of the bitter taste receptor. For example, polymorphisms in the hTAS2R38 (a receptor for Phenylthiourea (PTC)) have been correlated with the ability to detect propylthiouracil (PROP) (Kim et al, "Positional cloning of the human qualitative trap under bitter taste to phenylthiocarbonate", Science 299: 1221. 1225, 2003; Wooding et al, 2004. certain polymorphisms in alleles of the hTAS2R43 gene render a person very sensitive to bitter taste of the natural botanical compounds aloin and aristolochic acid The presence of bitter taste receptors or allelic variants or polymorphisms thereof or non-naturally occurring cell lines having one or more mutations (e.g., random mutations or site-specific mutations) of mutated forms thereof are all within the scope of the invention.

In certain embodiments, the nucleic acid is a fragment of a nucleic acid sequence provided above. Such bitter taste receptors as fragments or with such modifications retain at least one biological property of the bitter taste receptor, e.g., its ability to increase free calcium within the cell. The invention includes cells and cell lines that stably express a nucleotide sequence encoding a bitter taste receptor, the nucleotide sequence having at least about 85% identity to the sequences disclosed herein. In certain embodiments, the sequence encoding the subunit is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical compared to the subunit sequence provided herein. The invention also includes cells and cell lines in which a nucleic acid encoding a bitter taste receptor hybridizes under stringent conditions to a nucleic acid encoding a corresponding bitter taste receptor provided herein.

In certain embodiments, the cell or cell line comprises a nucleic acid sequence encoding a bitter taste receptor comprising at least one but less than 10, 20, 30, or 40 nucleotides compared to a sequence provided herein, up to or equal to 1%, 5%, 10%, or 20% substitution of the nucleotide sequence or from a sequence substantially identical thereto (e.g., a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity thereto, or a sequence capable of hybridizing to a disclosed sequence under stringent conditions). In certain embodiments, the cell or cell line comprises a nucleic acid sequence encoding a bitter taste receptor comprising less than 10, 20, 30, or 40 nucleotides, up to or equal to 1%, 5%, 10%, or 20% insertion or deletion of the nucleotide sequence or a sequence substantially identical thereto (e.g., a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto, or a sequence capable of hybridizing to a disclosed sequence under stringent conditions) as compared to a sequence provided herein. Substitutions, insertions, and deletions described herein may be present in any of the polynucleotides encoding bitter taste receptors of a cell or cell line of the invention.

In certain embodiments, where a nucleic acid substitution or modification results in an amino acid change, e.g., an amino acid substitution, the natural amino acid may be replaced with a conservative or non-conservative substitution. In certain embodiments, the sequence identity between the original and modified polypeptide sequences may differ by about 1%, 5%, 10%, or 20% of the polypeptide sequence or differ from a sequence substantially identical thereto (e.g., a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto). One of ordinary skill in the art will appreciate that a conservative amino acid substitution is one in which the amino acid side chains are similar in structure and/or chemical properties, and the substitution should not significantly alter the structural characteristics of the parent sequence. In embodiments that include nucleic acids comprising mutations, the mutations can be random mutations or site-specific mutations.

Conservative modifications will result in bitter taste receptors that have functional and chemical characteristics similar to those of the unmodified bitter taste receptor. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain R group of similar chemical properties (e.g., charge or hydrophobicity) as the parent amino acid residue. Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the degree of percent sequence identity or similarity may be adjusted upward to correct for the conservative nature of the substitution. Methods for making this adjustment are well known to those of ordinary skill in the art. See, e.g., Pearson, methodsfol. biol. 243: 307-31(1994).

Examples of groups of amino acids possessing side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy side chain: serine and threonine; 3) amide-containing side chain: asparagine and glutamine; 4) aromatic side chain: phenylalanine, tyrosine and tryptophan; 5) basic side chain: lysine, arginine and histidine; 6) acidic side chain: aspartic acid and glutamic acid; and 7) sulfur containing side chains: cysteine and methionine. Preferred conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate and asparagine-glutamine. Alternatively, conservative amino acid substitutions are described in Gonnet et al, Science 256: any change in the PAM250 log likelihood matrix with positive values as disclosed in 1443-45 (1992). A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log likelihood matrix.

In certain embodiments, the nucleic acid sequence encoding a bitter taste receptor further comprises a tag. Such tags may encode, for example, a HIS tag, a myc tag, a Hemagglutinin (HA) tag, proteins C, VSV-G, FLU, Yellow Fluorescent Protein (YFP), green fluorescent protein, FLAG, BCCP, a maltose binding protein tag, a Nus-tag, Softag-1, Softag-2, a Strep-tag, an S-tag, thioredoxin, GST, V5, TAP, or CBP. The tags can be used to determine bitter taste receptor expression levels, intracellular localization, protein-protein interactions, modulation of bitter taste receptors, or bitter taste receptor function. Tags may also be used to purify or fractionate taste receptors. In certain embodiments, the tag is cleavable from its labeled bitter taste receptor. This can be achieved, for example, by introducing a specific protease cleavage site between the tag and the bitter taste receptor in a nucleic acid encoding the bitter taste receptor and then subjecting the labeled bitter taste receptor expressed from the nucleic acid to treatment with the specific protease.

The host cells used to produce the cells or cell lines of the invention may express one or more endogenous bitter taste receptors in their native state or lack the expression of any bitter taste receptors. In the case where the cell or cell line expresses one or more of its own bitter taste receptors (also referred to as "endogenous" bitter taste receptors), the heterologous bitter taste receptor may be the same as one of the endogenous bitter taste receptors of the cell or cell line. For example, a nucleic acid encoding a bitter taste receptor endogenous to the cell or cell line can be introduced into the cell or cell line to increase the copy of the gene encoding the bitter taste receptor in the cell or cell line such that the bitter taste receptor is expressed at a higher level in the cell or cell line than in the absence of the introduced nucleic acid. The host cell may be a primary cell, a sperm cell or a stem cell, including an embryonic stem cell. The host cell may also be an immortalized cell. The primary or immortalized host cell may be derived from the mesoderm, ectoderm or endoderm of a eukaryote. The host cell may be an epithelial cell, an epidermal cell, a mesenchymal cell, a neural cell, a renal cell, a hepatic cell, a hematopoietic cell, or an immune cell. For example, the host cell may be an intestinal crypt or villus cell, a Clara cell, a colon cell, an intestinal cell, a goblet cell, an enterochromaffin cell, an enteroendocrine cell. The host cell can be a eukaryotic cell, prokaryotic cell, mammalian cell, human cell, primate cell, bovine cell, porcine cell, feline cell, rodent cell, marsupial cell, murine cell, or other cell. The host cell may also be a non-mammalian cell, such as yeast, insect, fungal, plant, lower eukaryote, and prokaryotic cells. Such host cells may provide a more diverse background for testing for bitter taste receptor modulators (with greater likelihood that the expression product provided by the cell that may interact with the target is absent). In a preferred embodiment, the host cell is a mammalian cell. Examples of host cells that can be used to generate the cells or cell lines of the invention include, but are not limited to: human embryonic kidney-293T cells, established neuronal cell lines, pheochromocytoma, neuroblastoma fibroblasts, rhabdomyosarcoma cells, dorsal root ganglion cells, NS0 cells, CV-1(ATCC CCL70), COS-1(ATCC CRL 1650), COS-7(ATCC CRL 1651), CHO-K1(ATCC CCL 61), 3T3(ATCC CCL 92), NIH/3T3(ATCC CRL1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCCCL 26), MRC-5(ATCC CCL 171), L-cells, HEK-293(ATCC CRL1573) and PC12(ATCC CRL 1721), HEK293T (ATCC CRL-11268), RBL (CCRL-1378), SH-SY 5(ATCC CRL-5Y), ATCC CRL-226K (ATCC CCL 22634), ATCC CRL 206861 (ATCC CRL 30), ATCC CRL 30 (SJ 861), HepG2(ATCC HB-8065), ND7/23(ECACC92090903), CHO (ECACC 85050302), Vero (ATCC CCL 81), Caco-2(ATCC HTB 37), K562(ATCC CCL 243), Jurkat (ATCCIB-152), Per.C6(Crucell, Leiden, The Netherlands), huvec (ATCC human primary PCS 100-010, mouse CRL 2514, CRL 2515, CRL 2516), HuH-7D12(ECACC 01042712), 293(ATCC CRL 10852), A549(ATCC CCCL 185), IMR-90(ATCC CCL 186), MCF-7(ATC HTB-22), U-2OS (ATCC HTB-96), T84(ATCC CCL 248) or any established cell line (polarized or non-polarized) or any cell line available from a library such as the American type culture Collection (ATCC, 10801University Blvd. assManas, Va.20110-2209USA) or the European cell culture Collection (ECACC, Salisbury Wiltshire SP40JG England).

In one embodiment, the host cell is an embryonic stem cell, which is then used as a basis for the production of transgenic animals. In certain embodiments, one or more bitter taste receptors may be expressed with temporal and/or tissue specific expression. Embryonic stem cells that stably express at least one bitter taste receptor, preferably a functional heterologous bitter taste receptor, can be directly implanted into an organism, or their nuclei can be transferred into other recipient cells, which can then be implanted, or they can be used to produce transgenic animals.

As will be appreciated by those of ordinary skill in the art, any vector suitable for use with the host cell of choice may be used to introduce the nucleic acid encoding the bitter taste receptor into the host cell. Examples of vectors that can be used to introduce nucleic acids into a host cell include, but are not limited to, plasmids, viruses including retroviruses and lentiviruses, cosmids, artificial chromosomes, which can include, for example, pCMVScript, pcDNA3.1Hygro, pcDNA3.1neo, pcDNA3.1puro, pSV2neo, pIRESpuro, pSV2 zeo. Exemplary mammalian expression vectors that can be used to generate the cells and cell lines of the invention include: pFN11A (BIND)pGL4.31、pFC14A(7)CMVpFC14K(7)CMVpFN24A(7)CMVd3pFN24K(7)CMVd3HaloTag^TMpHT2、pACT、pAdVAntage^TM、pBIND、An enhancer,Promoter, pCI, pCMVTNT ^TM、pG5luc、pSI、pTARGET^TM、pTNT^TM、pF12A RMpF12K RMpReg neo，pYES2/GS、pAd/CMV/V5-DESTVector, pAd/PL-DEST^TMA carrier,pDEST^TM27 carrier, a,pEF-DEST51 vector,pcDNA^TMDEST47 vector, pCMV/Bsd vector, pEF6/His A, B and C, pcDNA^TM2-DEST, pLenti6/TR, pLP-AcGFP1-C, pLPS-AcGFP1-N, pLP-IRESNeo, pLP-TRE2, pLP-RevTRE, pLP-LNCX, pLP-CMV-HA, pLP-CMV-Myc, pLP-RetroQ and pLP-CMVneo. Examples of vectors that may be used to introduce nucleic acids encoding bitter taste receptors into host cells include, but are not limited to, plasmids, viruses (including retroviruses and lentiviruses), cosmids, artificial chromosomes that may include, for example, pCMVScript, pcDNA3.1Hygro, pcDNA3.1neo, pcDNA3.1puro,pSV2neo、pIRES puro、pSV2zeo、pFN11A(BIND)pGL4.31、pFC14A(7)CMVpFC14K(7)CMVpFN24A(7)CMVd3pFN24K(7)CMVd3HaloTag^TM pHT2、pACT、pAdVAntage^TM、pBIND、comparison, comparison,An enhancer,Promoter, pCI, pCMVTNT^TM、pG5luc、pSI、pTARGET^TM、pTNT^TM、pF12A RMpF12K RMpRegneo、pYES2/GS、Vector, pAckPL-DEST^TMA carrier,pDEST^TM27 carrier, a,pEF-DEST51 vector,pcDNA^TMDEST47 vector, pCMV/Bsd vector, pEF6/HisA, B,& C、pcDNA^TM2-DEST, pLenti6/TR, pLP-AcGFP1-C, pLPS-AcGFP1-N, pLP-IRESNeo, pLP-TRE2, pLP-RevTRE, pLP-LNCX, pLP-CMV-HA, pLP-CMV-Myc, pLP-RetroQ, pLP-CMVneo. In certain embodiments, the vector comprises an expression control sequence such as a constitutive or conditional promoter, preferably a constitutive promoter is used. One of ordinary skill in the art will be able to select such sequences. For example, suitable promoters include, but are not limited to, CMV, TK, EF-1 α. In certain embodiments, the promoter is inducible, temperature regulated, tissue specific, repressible, heat shock, developmental, cell lineage specific, transient promoter or unmodified or mutagenized, randomized, shuffled of any one or more of the above Combinations or recombinations of sequences. In other embodiments, the bitter taste receptor is expressed by gene activation or when the gene encoding the bitter taste receptor is episomal. Preferably constitutively expressing an RNA encoding a bitter taste receptor or a mutant form or naturally occurring allelic variant thereof.

In certain embodiments, the vector lacks a selectable marker or drug resistance gene. In other embodiments, the vector optionally comprises a nucleic acid encoding a selectable marker (e.g., a protein that confers drug or antibiotic resistance). Selection pressure can be used in cell culture to select for cells having a desired sequence or trait, and this is typically achieved by combining expression of the polypeptide of interest with expression of a selectable marker that confers resistance to the corresponding selection agent or pressure on the cell. Antibiotic selection includes, but is not limited to, the use of antibiotics (e.g., puromycin, neomycin, G418, hygromycin, bleomycin, etc.). Non-antibiotic selection includes, but is not limited to, the use of nutrient deprivation, exposure to selection temperatures, and exposure to mutagenic conditions, where the selectable marker can be, for example, glutamine synthetase, dihydrofolate reductase (DHFR), oagain, Thymidine Kinase (TK), hypoxanthine guanine phosphoribosyl transferase (HGPRT). Each vector encoding a sequence for a different bitter taste receptor may have the same or different drug resistance or other selectable marker. If more than one drug resistance marker is the same, simultaneous selection can be achieved by increasing the level of drug. Suitable markers are well known to those of ordinary skill in the art and include, but are not limited to, genes that confer resistance to any of the following: neomycin/G418, puromycin, hygromycin, bleomycin, methotrexate and blasticidin. Although drug selection (or selection using any other suitable selectable marker) is not a necessary step, it can be used to enrich stably transfected cells from a transfected cell population, provided that the transfected construct is designed to confer drug resistance. If subsequent selection of cells expressing bitter taste receptors is performed using signaling probes, premature selection after transfection may result in some positive cells that may be only transiently transfected, but not stably transfected. However, this can be minimized by passaging sufficient cells to allow transient expression in the diluted transfected cells.

In certain embodiments, the vector comprises a nucleic acid sequence encoding an RNA tag sequence. "tag sequence" refers to a nucleic acid sequence that is an expressed RNA or portion of an RNA to be detected by a signaling probe. The signaling probe can detect a variety of RNA sequences. Any of these RNAs can be used as a tag. The signaling probe may be directed to the tag by designing the probe to include a portion complementary to the sequence of the tag. In particular embodiments, the signaling probes are directed to the same or different coding exons, non-coding introns, or non-coding untranslated sequences of (e.g., complementary) RNAs. In particular embodiments, the signaling probe can be directed to RNA of a component of a signaling pathway, including a bitter receptor. The tag sequence may be the 3' untranslated region of a plasmid that is co-transcribed and contains the target sequence to which the signaling probe binds. The RNA encoding the gene of interest may include a tag sequence or the tag sequence may be located in the 5 'untranslated region or the 3' untranslated region. In certain embodiments, the tag is not associated with an RNA encoding the gene of interest. The tag sequence may be present in-frame with the protein-coding portion of the messenger of the gene or not, depending on whether it is desired to tag the protein produced. Thus, translation of the tag sequence is not necessary for detection by the signaling probe. The tag sequence may comprise multiple target sequences, which may be the same or different, with one signaling probe hybridizing to each target sequence. The tag sequence may encode an RNA having secondary structure. The structure may be a three-arm connection structure. Examples of tag sequences that can be used in the present invention and for which signaling probes can be prepared include, but are not limited to, RNA transcripts of epitope tags such as HIS tags, myc tags, Hemagglutinin (HA) tags, proteins C, VSV-G, FLU, Yellow Fluorescent Protein (YFP), green fluorescent protein, FLAG, BCCP, maltose binding protein tags, Nus-tags, Softag-1, Softag-2, Strep-tags, S-tags, thioredoxin, GST, V5, TAP, or CBP. As described herein, one of ordinary skill in the art can generate his or her own RNA tag sequences.

In another aspect, the cells and cell lines of the invention stably express a G protein. There are two families of G proteins, heterotrimeric and monomeric G proteins. Heterotrimeric G proteins are activated by G protein-coupled receptors ("GPCRs"), which comprise 3 subunits: g_α、G_βAnd G_Y. As used herein, the term G protein includes any of such subunits, e.g., G_αOr any combination thereof and heterotrimeric G proteins having all 3 subunits. In the inactive state, G_α、G_βAnd G_YA trimer is formed. The beta and gamma subunits bind tightly to each other and are called beta-gamma complexes. G after ligand binding to GPCR_αAnd G_βYAnd (5) separating. G_βYFrom G after GDP-GTP exchange thereof_αThe subunits are released. G_βYThe complex may activate other second messengers or gated ion channels. The 4 families of ga include: g increasing cAMP Synthesis by activation of adenylate cyclase_s(irritancy); inhibition of adenylate cyclase G_i(inhibitory); g_12/13Families regulate a variety of cellular motor processes (i.e., cytoskeleton, cell junctions); and G_qWhich stimulates calcium signaling and phosphatase C. Monomeric G proteins are homologous to the alpha subunit of heterotrimeric G proteins. Any G protein can be expressed in the cells or cell lines of the invention, including, but not limited to, transducins (e.g., GNAT1, GNAT2, and guanine nucleotide binding protein G (t)), gustducins (e.g., GNAT3 guanine nucleotide binding protein and α transducin 3), human GNA15 (guanine nucleotide binding protein (G protein) α 15 (class Gq; synonyms GNA16), and mouse G α 15, as well as chimeric proteins thereof such as G α 15-GNA15, also known as G α 15-G α 16). In a preferred embodiment, the G protein is mouse G.alpha.15 (SEQ ID NO: 102). In another preferred embodiment, the G protein is human GNA15(SEQ ID NO: 50) or is encoded by a sequence comprising SEQ ID NO: 76, or a human G protein encoded by the nucleic acid of seq id no. The G protein can also be any mammalian G protein such as, but not limited to, any of the mammalian G proteins listed in table 7. The G protein stably expressed by the cell may be endogenous to the cell. Alternatively, Stable expression of a G protein can be the result of stable transfection of a nucleic acid encoding the G protein into a cell. Cells that stably express heterologous G proteins are known in The art, for example HEK 293/G.alpha.15 cells (Chandrashikar et al, "T2 Rsfunction as bit holder receptors", Cell 100: 703-711, 2000; Bufe et al, "The human TAS2R16 receptors bits in response to beta-glucanides", Na Genet 32: 397-401). In other embodiments, the nucleic acid encoding the G protein and the nucleic acid encoding the bitter taste receptor can be transfected into the host cell sequentially, either first with the nucleic acid encoding the G protein or first with the nucleic acid encoding the bitter taste receptor. In other embodiments, the nucleic acid encoding the G protein and the nucleic acid encoding the bitter taste receptor may be co-transfected into the host cell on the same or different vectors. Thus, selection of cells stably expressing the G protein and the bitter taste receptor can likewise be performed sequentially or simultaneously. The cells or cell lines that can be used to stably express the G protein are the same as the cells or cell lines that can be used to stably express the bitter taste receptor, as explained above. The vector used to transfect the G protein optionally comprises a nucleic acid encoding a selectable marker selected for antibiotic or non-antibiotic, as explained above.

In some embodiments of the invention, the cells or cell lines of the invention co-express other proteins with the bitter taste receptor. In a preferred embodiment, the additional protein is at least one additional taste receptor such as a sweet (TAS1R2/TAS1R3) receptor or an umami (TAS1R1/TAS1R3) receptor. The protein co-expressed with the bitter taste receptor may be expressed by any mechanism, such as, but not limited to, endogenously in the host cell or exogenously from the vector. Likewise, in other embodiments of the invention, more than one type of bitter taste receptor may be stably expressed in a cell or cell line.

In another aspect, the cells and cell lines of the invention have enhanced stability compared to cells and cell lines produced by conventional methods. To identify stable expression, the expression of bitter taste receptors in a cell or cell line is measured over a period of time and the expression levels are then compared. A stable cell line will continue to express bitter taste receptors continuously throughout the time course. In some aspects of the invention, the time course may last for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, etc., or at least 1 month or at least 2, 3, 4, 5, 6, 7, 8, or 9 months or any length of time therebetween. Isolated cells and cell lines can also be characterized, for example, by qRT-PCR and single-endpoint RT-PCR to determine the absolute and relative amounts of bitter taste receptor expressed. In certain embodiments, stable expression is measured by comparing binding of functional assays over a period of time. Measurement of stability based functional assays provides the benefit of identifying clones that stably express not only the mRNA of the gene of interest but also stably produce and correctly process (e.g., post-translational modifications, subunit assembly, and intracellular localization) the protein encoded by the gene of interest that functions correctly.

The cells and cell lines of the invention have the further advantageous property of providing assays with high reproducibility, as evidenced by their Z' factor. See Zhang JH, Chung TD, OldenburgKR, "a Simple statistical parameter for Use in evaluation and ValidationofHigh through screening assays," j.biomol. screen.1999; 4(2): 67-73, which references are incorporated herein by reference in their entirety. The Z' value relates to the quality of the cell or cell line as it reflects the extent to which the cell or cell line will respond consistently to the modulator. Z' is a statistical calculation that takes into account the range of signal-to-noise ratios and signal variability (i.e., from well to well) across the functional response of the multi-well plate to the reference compound. Z' was calculated using data from multiple wells with positive controls and multiple wells with negative controls. The ratio of the standard deviations of their combination is multiplied by 3 to the difference factor and the average is subtracted by 1 to obtain the Z' factor according to the following equation:

z' factor ═ 1- ((3 δ positive control +3 δ negative control)/(μ positive control- μ negative control))

The theoretical maximum Z' factor is 1.0, which would indicate an ideal determination of no variability and infinite dynamic range. Lower scores (i.e., scores near 0) are undesirable because they indicate an overlap between the positive and negative controls. In the industry, for simple cell-based assays, a Z ' score of up to 0.3 is considered an edge score, a Z ' score of 0.3 to 0.5 is indicated as acceptable, and a Z ' score exceeding 0.5 is indicated as excellent. Cell-free or biochemical assays can approach scores for cell-based systems, which tend to be lower because of higher Z 'scores, but Z' cell-based systems are complex.

The cells and cell lines of the invention have a Z' value that reflects their ability to advantageously produce consistent results in an assay. The bitter taste receptor expressing cells and cell lines of the present invention provide the basis for High Throughput Screening (HTS) compatibility assays, as they typically have a Z' value of at least 0.45. In some aspects of the invention, the cells and cell lines result in a Z' of at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, or at least 0.8. In other aspects of the invention, the cells and cell lines of the invention result in a Z' of at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, or at least 0.8 for a plurality of generations, e.g., 5 to 20 generations, including any integer number between 5 and 20 generations. In some aspects of the invention, the cells and cell lines result in a Z' of at least 0.4, at least 0.5, at least 0.6, at least 0.7, or at least 0.8 for 1, 2, 3, 4, or 5 weeks or 2, 3, 4, 5, 6, 7, 8, or 9 months, including any time period therebetween.

Also in accordance with the present invention, cells and cell lines expressing a form of a naturally occurring bitter taste receptor or naturally occurring allelic variant thereof, as well as cells and cell lines expressing a mutant form of a bitter taste receptor, can be characterized with respect to intracellular free calcium levels. In certain embodiments, the cells and cell lines of the invention express bitter taste receptors having "physiologically relevant" activity. As used herein, physiological relevance refers to the properties of a cell or cell line that expresses a bitter taste receptor that causes an increase in intracellular free calcium (as caused by the same type of naturally occurring bitter taste receptor when activated) and responds to a modulator in the same manner as the same type of naturally occurring bitter taste receptor responds when modulated by the same compound. The bitter taste receptor-expressing cells and cell lines of the invention, including some mutant forms of the bitter taste receptor and some naturally occurring allelic variants of the bitter taste receptor, preferably exhibit comparable function to cells that normally express the natural bitter taste receptor in appropriate assays, e.g., assays that measure intracellular free calcium. Such assays are known to those of ordinary skill in the art (Nahorski, "pharmacy of intracellular signaling pathwaters", Brit. J. Pharm. 147: S38-S45, 2000)). Such comparisons are used to determine the physiological relevance of a cell or cell line. A "sipand-spit" taste test using a panel of trained taste testers can also be used to further confirm the physiological relevance of the bitter taste receptors in the cells and cell lines of the invention. The results of a sniff taste test using modulators identified by screening for either the natural or mutated forms of bitter taste receptors or their naturally occurring allelic variants can be used to confirm the physiological relevance of these different forms.

In certain embodiments, the cells and cell lines of the invention have increased sensitivity to modulators of bitter taste receptors. The cells and cell lines of the invention respond to modulators and increase intracellular free calcium with physiological range EC50 or IC50 values for bitter taste receptors. As used herein, EC50 refers to the concentration of a compound or substance required to induce a hemimaximal activation response in a cell or cell line. As used herein, IC50 refers to the concentration of a compound or substance required to induce a half-maximal inhibitory response in a cell or cell line. EC50 and IC50 values can be determined using techniques well known in the art, such as dose-response curves relating the concentration of a compound or substance to the response of cell lines expressing bitter taste receptors.

Other advantageous properties of the bitter taste receptor-expressing cells and cell lines of the present invention that result from physiologically relevant functions of bitter taste receptors are that the modulators identified in the primary screen are functional in secondary functional assays such as an emetic or other taste test or functional Magnetic Resonance Imaging (MRI) that scans brain activity in response to taste modulating compounds. As will be appreciated by those of ordinary skill in the art, the compounds identified in the initial screening assay must generally be modified, e.g., by combinatorial chemistry, pharmaceutical chemistry, or synthetic chemistry, for their derivatives or analogs to function in a secondary functional assay. However, due to the high physiological relevance of cells and cell lines expressing the present bitter taste receptor, many of the compounds identified thereby are functional without modification.

In certain embodiments, properties of the cells and cell lines of the invention, such as stability, physiological relevance, reproducibility of determination (Z'), or physiological EC50 or IC50 values, are obtainable under specific culture conditions. In certain embodiments, culture conditions can be standardized and maintained strictly, e.g., by automation. The culture conditions may comprise any suitable culture under which the cells or cell lines are cultured, which may comprise conditions known in the art. Various culture conditions may result in favorable biological properties for any bitter taste receptor or mutant or allelic variant thereof.

In other embodiments, the cells and cell lines of the invention having desired properties such as stability, physiological relevance, reproducibility of assay (Z'), or physiological EC50 or IC50 values can be obtained in a month or less. For example, the cells or cell lines may be obtained within 2, 3, 4, 5, or 6 days, or within 1, 2, 3, or 4 weeks, or any length of time therebetween.

One aspect of the invention provides a collection of clonal cells and cell lines that each express the same bitter taste receptor or a different bitter taste receptor, including different mutant forms and naturally occurring allelic variants of one or more bitter taste receptors. The collection may include, for example, cells or cell lines that express a combination of different bitter taste receptors or a mutant form of a bitter taste receptor, a naturally occurring allelic variant of a bitter taste receptor, or any combination thereof. The collection may also include cells or cell lines expressing any possible dimers or other multimers of the same bitter taste receptor and different G proteins or bitter taste receptors, including heteromultimers or chimeric dimers or multimers.

When generating a collection or subject group of cells or cell lines, e.g., for drug screening, the cells or cell lines in the collection or subject group may be matched such that they are identical (including substantially identical) with respect to one or more selected physiological properties. By "the same physiological property" in this context is meant that the selected physiological property is sufficiently similar among the members of the collection or group of subjects that the collection of cells or group of subjects can produce reliable results in a drug screening assay; for example, differences in readings in a drug screening assay will be due to, for example, different biological activities of the test compound on cells expressing different native or mutated forms of the bitter taste receptor or allelic variants thereof, rather than due to inherent differences in the cells. For example, cells or cell lines may be matched to have the same growth rate, i.e., a growth rate that does not differ by more than 1, 2, 3, 4, or 5 hours among the members of a collection of cells or a group of subjects. This can be achieved, for example, by combining the cell frames into 5, 6, 7, 8, 9 or 10 groups via their growth rates, and generating a subject group using cells from the same combined frame and group. Methods for measuring cell growth rate are well known in the art. The cells or cell lines in the subject group can also be matched to have the same Z 'factor (e.g., a Z' factor that differs by no more than 0.1), bitter taste receptor expression levels (e.g., bitter taste receptor expression levels that differ by no more than 5%, 10%, 15%, 20%, 25%, or 30%), adhesion to tissue culture surfaces, and the like. Matched cells and cell lines can be grown under equivalent conditions, achieved by, for example, automated parallel processing, to maintain selected physiological properties.

The matched set of cell subjects of the invention can be used, for example, to identify modulators (e.g., agonists or antagonists) having a defined activity for bitter taste receptors; characterizing the activity of the compound differently between bitter taste receptors or allelic variants or mutant forms thereof; identifying modulators that are active only on one type of bitter taste receptor or allelic variants or mutant forms thereof; and identifying modulators that are active on only a subset of bitter taste receptors. The matched cell panel of the present invention allows for high throughput screening. Screening that takes months to complete can now be completed in weeks.

For the preparation of the cells and cell lines of the invention, techniques such as those described in U.S. Pat. No. 6,692,965 and International patent publication WO/2005/079462 may be used. Both of these documents are incorporated by reference herein in their entirety for all purposes. This technique provides a real-time assessment of millions of cells, thereby allowing the detection of any desired number of clones (from hundreds to thousands of clones). Using cell sorting techniques, such as flow cytometry cell sorting (e.g., with a FACS machine) or magnetic cell sorting (e.g., with a MACS machine), 1 cell/well can be automatically deposited in culture vessels (e.g., 96-well culture plates) with high statistical reliability. The speed and automation of the technology allows for easy isolation of multigene recombinant cell lines.

By using the described techniques, the RNA sequence of each bitter taste receptor can be detected using signaling probes (also known as molecular beacons or fluorescent probes). In certain embodiments, the molecular beacon recognizes a target tag as described above. In another embodiment, the molecular beacon recognizes a sequence within the bitter taste receptor coding sequence itself. The signaling probe can be directed to the RNA tag or bitter taste receptor coding sequence by designing the probe to include a portion complementary to the RNA sequence of the tag or bitter taste receptor coding sequence, respectively. Such same techniques can be used to detect the RNA sequence of the G protein (if used).

Nucleic acids comprising a sequence encoding a bitter taste receptor, a sequence encoding a G protein, a tag sequence, or any combination thereof, and optionally further comprising a nucleic acid encoding a selectable marker, can be introduced into a selected host cell using well known methods. Techniques for introducing nucleic acids into cells are well known and can be readily understood by one of ordinary skill in the art. Such methods include, but are not limited to, transfection, viral delivery, protein or peptide mediated insertion, co-precipitation methods, lipid-based delivery reagents (lipofection), cytofectins, lipopolyamine delivery, dendrimer delivery, electroporation, or mechanical delivery. Examples of transfection agents are GENEPORTER, GENEPORTER2, LIPOFECTANCE 2000, FUGENENE 6, FUGENENE HD, TFX-10, TFX-20, TFX-50, OLIGOFECTAMINE, TRANSFAST, TRANSFECTAM, GENESHUTTLE, TROXENE, GENESILENCER, X-TREMEGENE, PERFECTIN, CYTOFECTIN, SIPORT, UNIFECTOR, SIFECTOR, TRANSIT-LT1, TRANSIT-LT2, TRANSIT-EXPRESS, IFECT, RNASITTLE, METAFACTENE, LYOVEC, LIPOTAXI, GENEERASER, GENEJUICE, CYTOPURE, TSJEI, JETPTPE, MEFEGACTIN, POFELYCT, TRANSMES SANGER, AIRNFEEI, SUPERENCE, EFCTENE, PEI-KIT, CLOCTIN, and CLONCTIN METAFECTINE.

After introduction of bitter taste receptor coding sequences and optionally also G protein coding sequences into host cells, optionally followed by drug selection, molecular beacons (e.g., fluorescent probes) are introduced into the cells and cell sorting is used to isolate cells positive for their signal. Molecular beacons may also be used to identify expression of bitter taste receptors, G proteins, or both. If the expression of both is identified, their identification can be performed simultaneously or sequentially. If necessary, multiple rounds of sorting can be performed. In one embodiment, the flow cytometric sorter is a FACS machine. MACS (magnetic cell sorting) or laser ablation of negative cells using laser activated analysis and treatment can also be used. According to the method, cells expressing at least one bitter taste receptor are detected and recovered. Bitter taste receptor (and optionally G protein) sequences can be integrated into the cell at various locations in the genome. The level of expression of the introduced gene encoding the bitter taste receptor (and, if introduced, the G protein) may vary based on the integration site. One of ordinary skill in the art will appreciate that sorting can be gated on a desired expression level (i.e., above background or at a particular level above background). Furthermore, stable cell lines can be obtained in which one or more of the introduced genes encoding bitter taste receptors or G proteins are episomal.

Signaling probes useful in the present invention are known in the art and are generally oligonucleotides comprising a sequence complementary to a target sequence and a signal emitting system arranged such that when the probe is not bound to the target sequence, no signal is emitted and when the probe is bound to the target sequence, a signal is emitted. By way of non-limiting example, a signaling probe may include a fluorophore and a quencher positioned in the probe such that the quencher and fluorophore are brought together in an unbound probe. Upon binding between the probe and target sequence, the quencher and fluorophore separate, resulting in emission of a signal. For example, international publication WO/2005/079462 describes a number of signaling probes that may be used in the generation of the cells and cell lines herein. The methods described above for introducing nucleic acids into cells can be used to introduce signaling probes. In the case of using a tag sequence, if a plurality of bitter taste receptors are expressed in one cell, the carrier for each bitter taste receptor or the carriers for the bitter taste receptor and the G protein may contain the same or different tag sequences. Whether the tag sequences are the same or different, the signaling probes may comprise different signaling emitters, e.g., fluorophores with different colors, etc., thereby allowing for the separate detection of the expression (RNA) of each different bitter taste receptor and optionally of the G protein. By way of illustration, a signaling probe that specifically detects a bitter taste receptor mRNA can comprise a red fluorophore, and a probe that detects an introduced G protein (RNA) can comprise a green fluorescent protein. Also by way of illustration, a signaling probe that specifically detects one bitter receptor mRNA can comprise a red fluorophore, a signaling probe that specifically detects another bitter receptor mRNA can comprise a green fluorescent protein group, and a signaling probe that specifically detects a third bitter receptor can comprise a yellow fluorophore. One of ordinary skill in the art will know of other methods for differentially detecting multiple bitter taste receptors or bitter taste receptors and G protein expression in cells transfected with multiple bitter taste receptors using signaling probes.

Nucleic acids encoding signaling probes can be introduced into a selected host cell by any of a number of methods well known to those of ordinary skill in the art, including, but not limited to, transfection, co-precipitation, lipid-based delivery reagents (lipofection), cytofectins, lipopolyamine delivery, dendrimer delivery, electroporation, or mechanical delivery. Examples of transfection agents are GENFPORTER, GENFPORTER2, LIPOFECTANF, LIPOFECTAMINE 2000, FUGENE6, FUGENE HD, TFX-10, TFX-20, TFX-50, OLIGOFECTAMINE, TRANSFAST, TRANSFECTAM, GENE SHUTTLE, TROJENE, GENE SILENCER, X-TREMEGENE, PERFECTIN, CYTOFECTIN, SIPORT, UNIFECTOR, SIFECTOR, TRANSIT-LT1, TRANSIT-LT2, TRANSIT-PREEXSS, IFECT, RNASHUTTLE, METAFACTLENE, LYOVEC, LIPOTAXI, GENEERASE, GENEJUICE, CYURE, TSMETPTAI, GAFECTIN, POLYFECT, TRANSMES SANGER, RNAAIFECT, SUPERENTE, FECTF, PEI-KIT, CLONFET, and METAFECTINE.

In one embodiment, the signaling probes are designed to be complementary to portions of the RNA encoding the protein of interest or to portions of their 5 'or 3' untranslated regions. Even if a signaling probe designed to recognize the messenger RNA of interest is capable of detecting falsely endogenously expressed target sequences, the ratio of these is such that the sorter can distinguish between 2 cell types, compared to the ratio of the sequence of interest produced by the transfected cells. Thus, constructs comprising nucleic acids encoding bitter taste receptors do not require and do not include sequences encoding fluorescent proteins. Thus, the heterologous fluorescent protein is not expressed in the cells of the invention. The cells or cell lines of the invention stably express heterologous native bitter taste receptors. While such protein tags/chaperones have been used for the expression of many bitter taste receptors to facilitate transport of the bitter taste receptors to the cell surface, the cells and cell lines of the present invention advantageously functionally express the bitter taste receptors on the surface of cells that do not have the novel bitter taste receptors.

In another embodiment of the invention, adherent cells may be adapted to suspension either before or after cell sorting and isolation of individual cells. In other embodiments, the isolated cells can be cultured individually or in mixtures to produce a population of cells. It is also possible to culture single or multiple cell lines separately or to culture the cells or cell lines in a mixture. If the mixture of cell lines produces the desired activity or has the desired properties, it can be further fractionated up to the identification of the cell line or group of cell lines having this effect. Mixing cells or cell lines may make it easier to maintain a large number of cell lines without having to maintain each cell line separately. Thus, for positive cells, a mixture of cells or cell lines can be enriched. The enriched mixed cells may be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% positive for the desired property or activity.

a) providing a plurality of cells expressing mRNA encoding a bitter taste receptor;

c) Culturing the cells under a desired set of culture conditions using an automated cell culture process characterized by substantially identical conditions for each isolated cell culture, normalizing the number of cells in each isolated cell culture during the culturing process, and wherein the isolated cultures are passaged according to the same protocol;

d) determining at least one desired characteristic of the bitter taste receptor of the isolated cell culture at least 2 times; and

According to this method, the cells are cultured under a desired set of culture conditions. The conditions may be any desired conditions. One of ordinary skill in the art will appreciate which parameters are included within a set of culture conditions. For example, culture conditions include, but are not limited to: culture medium (basal medium (DMEM, MEM, RPMI, serum-free, serum-containing, complete chemical composition-limited, animal-derived free component), monovalent and divalent ion (sodium, potassium, calcium, magnesium) concentrations, with addition of additionalExternal components (amino acids, antibiotics, glutamine, glucose or other carbon sources, HEPES, channel blockers, modulators of other targets, vitamins, trace elements, heavy metals, cofactors, growth factors, anti-apoptotic agents), fresh or conditioned media, with HEPES, pH, depletion or limitation of specific nutrients (amino acids, carbon sources)), confluent levels that the cells are allowed to reach before division/passage ₂Three gas systems (oxygen, nitrogen, carbon dioxide), humidity, temperature, resting or using a shaker, etc., as will be well known to those of ordinary skill in the art.

In certain embodiments, the method comprises the additional step of measuring the growth rate of the isolated cell culture. The growth rate can be determined using any of a variety of techniques well known to the skilled artisan. Such techniques include, but are not limited to, measuring ATP, cell confluence, light scattering, optical density (e.g., OD 260 for DNA). Preferably, the growth rate is determined using a method that minimizes the amount of time the culture spends outside of the selected culture conditions.

In certain embodiments, cell confluence is measured and growth rate is calculated from the confluence value. In certain embodiments, cells are dispersed and clots are removed for improved accuracy prior to measuring cell confluence. Methods for making cells monodisperse are well known and can be achieved, for example, by adding a dispersing agent to the culture to be measured. Dispersants are well known and readily available and include, but are not limited to, enzymatic dispersants such as trypsin, and EDTA-based dispersants. Growth rates can be calculated from the confluent date using commercially available software for this purpose, such as HAMILTON VECTOR. Automated confluence measurements, for example using automated microplate readers, are particularly useful. Plate readers for measuring confluence are commercially available and include, but are not limited to, CLONE SELECTIMAGER (Genetix). Typically, at least 2 measurements of cell confluence are made before calculating the growth rate. The number of confluent values used to determine growth rate may be any number convenient or suitable for culture. For example, confluence may be measured multiple times over, for example, 1 week, 2 weeks, 3 weeks, or any time period and at any desired frequency.

In step d), cells and cell lines may be tested and selected according to any physiological property including, but not limited to: an alteration in a cellular process encoded by the genome; alterations in cellular processes regulated by the genome; a change in a pattern of chromosome activity; a change in a pattern of chromosome silencing; a change in gene silencing pattern; a change in gene activation pattern or efficiency; a change in gene expression pattern or efficiency; alteration of RNA expression pattern or efficiency; changes in RNAi expression pattern or efficiency; alteration of RNA processing pattern or efficiency; alteration in RNA transport pattern or efficiency; a change in protein translation pattern or efficiency; a change in protein folding pattern or efficiency; a change in protein assembly pattern or efficiency; a change in the pattern or efficiency of protein modification; a change in protein transport pattern or efficiency; a change in the mode or efficiency of transport of membrane proteins to the cell surface; a change in growth rate; a change in cell size; a change in cell shape; a change in cell morphology; a change in% RNA content; a change in% protein content; change in% moisture content; changes in% lipid content; a change in ribosome content; a change in mitochondrial content; a change in ER quality; a change in plasma membrane surface area; a change in cell volume; alterations in the lipid composition of the plasma membrane; alteration of lipid composition of the nuclear envelope; changes in the protein composition of the plasma membrane; alteration of the protein composition of the nuclear envelope; a change in the number of secretory vesicles; (ii) a change in the number of lysosomes; a change in the number of cavitation bubbles; alterations in the cells with respect to the following abilities or potentials: protein production, protein secretion, protein folding, protein assembly, protein modification, enzymatic modification of proteins, protein glycosylation, protein phosphorylation, protein dephosphorylation, metabolite biosynthesis, lipid biosynthesis, DNA synthesis, RNA synthesis, protein synthesis, nutrient uptake, cell growth, mitosis, meiosis, cell division, dedifferentiation, conversion into stem cells, conversion into pluripotent cells, conversion into totipotent cells, conversion into stem cell types of any organ (i.e., liver, lung, skin, muscle, pancreas, brain, testis, ovary, blood, immune system, nervous system, bone, cardiovascular system, central nervous system, gastrointestinal tract, stomach, thyroid, tongue, gall bladder, kidney, nose, eye, toenail, hair, taste bud), conversion into any cell type that is differentiated (i.e., muscle, toenail, hair, taste bud), Cardiac muscle, neurons, skin, pancreas, blood, immune, red blood cells, white blood cells, killer T cells, enteroendocrine cells, taste, secretory cells, kidney, epithelial cells, endothelial cells, and also including any of the enumerated animal or human cell types that may be used to introduce nucleic acid sequences), uptake DNA, uptake small molecules, uptake fluorescent probes, uptake RNA, attachment to solid surfaces, adaptation to serum-free conditions, adaptation to serum-free suspension conditions, adaptation to scaled-up cell culture, use in large-scale cell culture, use in drug development, use in high-throughput screening, use in functional cell-based assays, use in membrane potential assays, use in calcium flow assays, use in G protein receptor assays, use in reporter cell-based assays, for use in an ELISA study, for use in an in vitro assay, for use in an in vivo application, for use in a secondary test, for use in a compound test, for use in a binding assay, for use in a panning assay, for use in an antibody panning assay, for use in an imaging assay, for use in a microimaging assay, for use in a multi-well plate, for adaptation to automated cell culture, for adaptation to miniaturized automated cell culture, for adaptation to large scale automated cell culture, for adaptation to cell culture in a multi-well plate (6, 12, 24, 48, 96, 384, 1536 or higher density), for use in a cell chip, for use on a slide, for use on a glass slide, for microarray on a slide or glass slide, for immunofluorescence studies, for use in protein purification, for use in bioproduct production, for use in industrial enzyme production, for use in reagent production for research, for use in vaccine development, for use in cell therapy, for use in transplants or humans, for use in factor isolation secreted by cells, for preparation of cDNA libraries, for purification of RNA, for purification of DNA, for infection by pathogens, viruses or other factors, for resistance to drugs, for suitability maintained under automated miniaturized cell culture bars, for use in protein production for characterization, comprising: protein crystallography, vaccine development, stimulation of the immune system, antibody production, or antibody production or testing. One of ordinary skill in the art will readily recognize suitable tests for any of the above listed characteristics. In particular embodiments, one or more such physical properties may be consistent physical properties associated with bitter taste receptors and may be used to monitor expression of functional bitter taste receptors.

Tests that may be used to characterize the cells and cell lines of the invention and/or the matched panel of subjects of the invention include, but are not limited to: amino acid analysis, DNA sequencing, protein sequencing, NMR, tests for protein transport, tests for nuclear mass transport, tests for subcellular localization of proteins, tests for subcellular localization of nucleic acids, microscopic analysis, sub-microscopic analysis, fluorescence microscopy, electron microscopy, confocal microscopy, laser ablation techniques, cell counting, and dialysis. The skilled person will know how to use any of the tests listed above.

In embodiments that include a step of measuring the growth rate, the cells are cultured for a time sufficient to adapt them to the culture conditions prior to measuring the growth rate. As the skilled artisan will appreciate, the length of time may vary depending on a number of factors, such as the cell type, the conditions selected, the culture format, and may be any amount of time from 1 day to several days, 1 week, or more.

Preferably, each individual culture of the plurality of isolated cell cultures is maintained under substantially equivalent conditions, including a standardized maintenance schedule, discussed below. Another advantageous feature of this method is that a large number of individual cultures can be maintained simultaneously, thereby allowing identification of cells with the desired trait group, even if very rare. For these and other reasons, according to the present invention, a plurality of isolated cell cultures are cultured using an automated cell culture method such that the conditions are substantially equivalent for each well. Automated cell culture prevents the inevitable variability inherent in artificial cell culture.

Any automated cell culture system can be used in the methods of the invention. Many automated systems are commercially available and well known to those of ordinary skill in the art. In certain embodiments, the automated system is a robotic system. Preferably, the system includes independently moving channels, a multi-channel head (e.g., 96-point head) and a clamp or preferential pick arm, and a HEPA filtration device to maintain sterility during operation. Number of channels in pipettor It should be suitable for the culture form. A convenient pipette has, for example, 96 or 384 channels. Such systems are known and commercially available. For example, MICROLAB STAR^TMAn instrument (Hamilton) can be used in the process of the invention. An automated system should be able to perform a variety of desired cell culture tasks. Such tasks will be known to those of ordinary skill in the art. They include, but are not limited to: removing culture medium, replacing culture medium, adding reagents, washing cells, removing wash solution, adding dispersants, removing cells from culture vessels, adding cells to culture vessels, and the like.

The generation of the cells or cell lines of the invention may comprise any number of isolated cell cultures. However, the advantages provided by the method increase as the number of cells increases. There is no theoretical upper limit on the number of cells or isolated cell cultures that can be utilized in the method. According to the invention, the number of isolated cell cultures may be 2 or more, but more advantageously is at least 3, 4, 5, 6, 7, 8, 9, 10 or more isolated cell cultures, such as at least 12, at least 15, at least 20, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 48, at least 50, at least 75, at least 96, at least 100, at least 200, at least 300, at least 384, at least 400, at least 500, at least 1000, at least 10,000, at least 100,000, at least 500,000 or more.

A further advantageous property of the cells and cell lines of the invention that express bitter taste receptors is that they stably express one or more bitter taste receptors in the absence of drug selection pressure. Thus, in a preferred embodiment, the cells and cell lines of the invention are maintained in the absence of any selective agent. In other embodiments, the cells and cell lines are maintained in the absence of any antibiotic. As used herein, cell maintenance refers to culturing the cells after they are selected for their bitter taste receptor expression as described above. Maintenance does not refer to an optional step of culturing the cells in a selection drug (e.g., an antibiotic) prior to cell sorting, wherein the introduction of a drug resistance marker into the cells allows for enrichment of stable transfectants in the mixed population.

Drug-free cell maintenance offers many advantages. For example, drug resistant cells do not always express the co-transfected transgene of interest at sufficient levels, as selection depends on the survival of cells that have taken up the drug resistance gene and contain or do not contain the transgene. In addition, the selection of drugs is often mutagenic or otherwise interferes with the physiology of the cell, resulting in biased results in cell-based assays. For example, selection of drugs can reduce susceptibility to apoptosis (Robinson et al, Biochemistry, 36 (37): 11169-11178(1997)), increase DNA repair and drug metabolism (Deffie et al, Cancer Res.48 (13): 3595-3602(1988)), increase cell pH (Thiebaut et al, J Histochem Cytomem 38.38 (5): 685 690 (1990); Roepe et al, biochemistry.32 (41): 11042-11056(1993)), Simon et al, Proc Natl Acad Sci U S A.91 (3): 1128-1132(1994)), decrease lysosome and endosome pH (Schinder et al, biochemistry.35 (9): 2811-2817 (1996)), Altan et al, J exp.187 (10): 1583): 1588): 1108 (1998)), decrease plasma membrane conductance (11032-ATP (11032) (Gi et al, 11041) (biochem et al, 11023, 1993)), increase plasma membrane conductance (Robinson et al, 11032) (ATP 76, 11032, 1993), proc NatlAcad Sci usa.90 (1): 312-: 4432-4437(1999)). Thus, the cells and cell lines of the invention allow screening assays that do not have any artifacts caused by the selection of drugs. In certain preferred embodiments, the cells and cell lines of the invention are not cultured with a selection pressure factor, such as an antibiotic, either before or after cell sorting, such that cells and cell lines having the desired properties are isolated by sorting even when not starting from an enriched cell population.

The expression level of bitter taste receptors may vary from cell to cell line. The expression level of a cell or cell line can also decrease over time due to epigenetic events such as DNA methylation and gene silencing, and loss of copies of the transgene. Such changes can be attributed to a number of factors such as the copy number of the transgene taken up by the cell, the genomic integration site of the transgene, and the integrity of the transgene after genomic integration. Expression levels can be estimated using FACS or other cell sorting methods (i.e., MACS). Additional rounds of introducing signaling probes can be used, for example, to determine whether and to what extent a cell remains positive over time for any one or more of the RNAs against which the cell was originally isolated.

The ease with which an isolation expressing all of the desired RNA can be re-isolated from cells that can no longer express all of the RNA of interest makes it possible to maintain the cell line in the absence of the drug or in the presence of a minimal concentration of the drug. The signaling probes may also be reused with previously generated cells or cell lines, for example, to determine whether or to what extent a cell remains positive for any one or more of the RNAs against which the cell was originally isolated.

In another aspect, the invention provides methods of using the cells and cell lines of the invention. The cells and cell lines of the invention may be used in any application where a functional bitter taste receptor is desired. The cells and cell lines can be used in, for example, but not limited to, in vitro cell-based assays or in vivo assays where cells are implanted into an animal (e.g., a non-human mammal) to, for example, screen bitter taste receptor modulators; assessing the bitterness of the substance; generating proteins for crystallography and binding studies; and studying the selectivity and dose of the compound, receptor/compound binding kinetics and stability, and the effects of receptor expression on cell physiology (e.g., electrophysiology, protein trafficking, protein folding, and protein regulation). The cells and cell lines of the invention may also be used in knock-down studies to study the effects of a particular bitter taste receptor or group of bitter taste receptors.

Cells and cell lines expressing various combinations of bitter taste receptors can be used separately or together to identify bitter taste receptor modulators, including modulators specific for a particular bitter taste receptor or mutant form or naturally occurring allelic variant of a bitter taste receptor, and to obtain information about the activity of each form.

Modulators include any substance or compound that alters the activity of a bitter taste receptor or a mutant form or naturally occurring allelic variant thereof. Modulators may be bitter taste receptor agonists (enhancers or activators) or antagonists (inhibitors or blockers), including partial agonists or antagonists, selective agonists or antagonists, and inverse agonists, and may be allosteric modulators. The substance or compound is a modulator, even if its modulating activity varies under different conditions or concentrations or with respect to different forms of the bitter taste receptor. In other aspects, a modulator can alter the ability of another modulator to affect the function of a bitter taste receptor. For example, a modulator in the form of a bitter taste receptor that is not inhibited by an antagonist may render that form of bitter taste receptor susceptible to inhibition by the antagonist.

The cells and cell lines of the invention can be used to identify the role of different forms of bitter taste receptors in different bitter taste receptor pathological states by correlating the identity of the in vivo form of the bitter taste receptor with the identity of known forms of bitter taste receptors based on their response to various modulators. This allows the selection of disease or tissue specific modulators for highly targeted treatment of such bitter taste receptor-associated pathologies or other physiological conditions. For example, because many naturally occurring bitter compounds are toxic, bitter receptors can be used as alarm sensors for ingestion of toxic food compounds. Bitter receptors expressed in the gastrointestinal mucosa may be involved in functional detection of nutrients and harmful substances in the lumen and prepare the intestine to absorb them or initiate a protective response. They may also be involved in the control of food intake by activating the gut-cranial nerve pathway. Thus, the bitter taste receptor modulators identified using the cell lines and methods of the present invention may be used to modulate nutrient absorption in a number of contexts, for example to control appetite and/or reduce nutrient absorption in the gut of obese persons, or to control muscle sensation and/or increase nutrient and/or energy absorption from food in malnourished persons. Bitter taste receptor modulators may also be used to identify bitter compounds, further characterize specific chemical or structural motifs or key residues of bitter taste receptors that affect binding properties, identify bitter taste receptors that are broadly, moderately, or selectively modulated for ligand binding, determine groups and subgroups of bitter taste receptors based on their binding profiles, deselect orphan bitter taste receptors, use these data for molecular modeling or drug design of bitter taste receptors and determine in which tissue various bitter taste receptors are active.

To identify modulators of bitter taste receptors, the novel cells or cell lines of the invention can be contacted with a test compound under conditions in which bitter taste receptors are expected to function, and then tested for a statistically significant change in bitter taste receptor activity (e.g., p < 0.05) as compared to an appropriate control (e.g., cells not contacted with the test compound). Positive and/or negative controls using known agonists or antagonists and/or cells expressing different bitter taste receptors or mutated forms or naturally occurring allelic variants thereof may also be used. In certain embodiments, the bitter taste receptor activity to be detected and/or measured is a change in intracellular free calcium levels. One of ordinary skill in the art will appreciate that various assay parameters (e.g., signal-to-noise ratio) may be optimized.

In other related aspects, the invention provides methods of identifying ligands for other GPCRs. Any GPCR may be used in the method, including but not limited to mammalian or human GPCRs as well as orphaned or deselected GPCRs. A non-limiting example of a desorbed GPCR is an opioid (listed in Table 9). Libraries of compounds or extracts can be used to screen cells or cell lines expressing GPCRs to generate expression profiles for the receptors. Receptors with similar profiles are grouped together and screened using compounds to identify ligands that bind the receptor.

In a further aspect, the invention provides methods of identifying ligands for orphan bitter taste receptors, i.e., the invention provides methods of deselecting bitter taste receptors. Libraries of compounds or extracts can be used to screen cells or cell lines expressing bitter taste receptors without known modulators to generate expression profiles of the receptors. Optionally, receptors with similar profiles (if any) are grouped together and screened using known bitter compounds to identify ligands that bind the receptor. Once the ligand is identified, the results can be further confirmed using taste testing. Advantageously, the cells and cell lines of the invention stably express native (i.e., unlabeled) bitter taste receptors, and thus the ligands identified using the present methods are accurate and relevant. In related embodiments, the method of deselecting bitter taste receptors may be used to deselect any orphan GPCR (including any orphan mammalian GPCR or any orphan human GPCR), such as those listed in table 8.

In certain embodiments, one or more cells or cell lines of the invention, including a collection of cell lines, can be contacted with a plurality of test compounds, e.g., a library of test compounds. The cell lines of the invention can be used to screen libraries of test compounds to identify one or more modulators. The test compound can be a chemical moiety, including a small molecule, polypeptide, peptide, peptidomimetic, antibody, or antigen-binding portion thereof. In the case of antibodies, they may be non-human antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies. The antibody can be an intact antibody comprising fully complementary heavy and light chains or an antigen-binding portion of any antibody, including antibody fragments (e.g., Fab and Fab, Fab ', F (ab') ₂、Fd、Fv、dAb, etc.), single chain antibodies (scFv), single domain antibodies, all or an antigen binding portion of a heavy or light chain variable region.

The cells or cell lines of the invention can also be used to generate sets with specific variables. For example, a collection may include: cells or cell lines that all express the same bitter taste receptor and different G proteins to study receptor-G protein interactions; or cells or cell lines expressing multiple endogenous and/or heterologous bitter taste receptors to study dimerization or formation of potential heteromultimers. The collection of cells or cell lines of the invention may also comprise in a preferred embodiment all 25 bitter taste receptors. Such a panel of subjects can be used to determine the on-target versus off-target activity of a compound, or the effect of a receptor in pure bitter versus related (i.e., astringent or metallic) taste.

The cells and cell lines of the invention can be used to identify participants in GPCR pathways other than the bitter taste receptors they stably express. For example, nucleic acids expressing different G proteins can be introduced (transiently or stably) into each cell line in a collection of cell lines expressing the same heterologous bitter taste receptor and preferably not expressing an endogenous bitter taste receptor. Any interaction between such G proteins and bitter taste receptors expressed by cell lines can be determined, for example, by detecting changes in intracellular free calcium upon contacting the cells with known bitter taste receptor agonists.

In certain embodiments, a large collection of compounds is tested for bitter taste receptor modulating activity in cell-based functional High Throughput Screening (HTS), for example, using 96-well, 384-well, 1536-well or higher density formats. In certain embodiments, more than one cell or cell line (including a collection of cell lines) of the invention can be used to screen a test compound or a plurality of test compounds, including a library of test compounds. If multiple cells or cell lines are used, each expressing a different naturally occurring or mutant bitter taste receptor molecule, then modulators that are effective against multiple bitter taste receptors or mutant forms or naturally occurring allelic variants thereof, or alternatively modulators that are specific for a particular bitter taste receptor or mutant form or naturally occurring allelic variant thereof and that do not modulate other bitter taste receptors or other forms of bitter taste receptors, can be identified. In the case of a cell or cell line of the invention that expresses a human bitter taste receptor, the cell can be contacted with a test compound to identify compounds that modulate bitter taste receptor activity (either increase or decrease) for use in treating a disease or condition characterized by an undesirable bitter taste receptor activity or a reduced desired bitter taste receptor activity or the absence of a desired bitter taste receptor activity.

In certain embodiments, the cells or cell lines of the invention may be modified by pretreatment with, for example, enzymes including mammalian or other animal enzymes, plant enzymes, bacterial enzymes, enzymes from lysed cells, protein modifying enzymes, lipid modifying enzymes, and enzymes in the oral cavity, gastrointestinal tract, stomach, or saliva prior to contact with the test compound. Such enzymes may include, for example, kinases, proteases, phosphatases, glycosidases, oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases, and the like. Alternatively, cells and cell lines can be first contacted with a test compound and then treated to identify compounds that alter the modification of the protein by the treatment.

In certain aspects, provided herein are methods that exploit the naturally occurring high degree of genetic diversity present in cells, and efficiently identify, select, and enrich for cells having a desired gene expression profile that confers a desired property (e.g., stable and/or high expression of a functional bitter taste receptor). The present methods can identify, select and enrich cells with improved properties from a collection of genetically diverse cells more quickly and efficiently than conventional methods. In particular embodiments, the cell is not genetically modified. In other specific embodiments, the methods allow for the generation of novel homogeneous populations of cells with improved properties (e.g., more stable and/or higher expression of functional bitter taste receptors).

In certain embodiments, the methods described herein comprise selecting a naturally occurring cell having one or more desired properties (e.g., stable and/or high expression of a bitter taste receptor). In particular embodiments, the methods described herein comprise selecting a cell that has a naturally occurring variant or mutation in one or more bitter taste receptor subunit genes.

In particular embodiments, the methods described herein comprise selecting an isolated cell that has a naturally occurring variant or mutation in the promoter region of a bitter taste receptor gene or a non-coding region (e.g., an intron, a 5 'untranslated region, and/or a 3' untranslated region) of a bitter taste receptor gene. Variants or mutations in the promoter region of a bitter taste receptor gene or in a non-coding region of a bitter taste receptor gene may result in higher and/or more stable expression of the gene product. In particular embodiments, the promoter region of the bitter taste receptor gene or a non-coding region of the bitter taste receptor gene has been modified, for example, by methylation or acetylation of the DNA. In particular embodiments, the cell comprises an epigenetic modification that affects chromatin remodeling with respect to a bitter taste receptor gene. Non-limiting examples of epigenetic modifications include, but are not limited to, ethenylation, methylation, ubiquitination, phosphorylation and phosphorylation, and ubiquitination.

In certain other embodiments, the invention comprises selecting cells that have undergone a pretreatment. Such pre-treatment may be exposure to sunlight or Ultraviolet (UV) light, mutagens such as EMS (ethyl methane sulfonate) and chemical agents. In particular embodiments, such pretreatment may include exposure to undesirable growth conditions, such as hypoxic or low nutrient conditions or toxic conditions.

The methods described herein provide for identifying and/or selecting cells (e.g., eukaryotic cells) that express one or more genes of interest (e.g., bitter taste receptor subunit genes). In certain embodiments, the gene of interest is expressed at a higher level than other cells as a result of genetic variability.

In particular embodiments, the methods described herein comprise (a) introducing into a cell (e.g., a eukaryotic cell) one or more signaling probes capable of detecting RNA of a bitter taste receptor; and (b) determining whether the cell (e.g., eukaryotic cell) comprises RNA of a bitter taste receptor. Such methods may also include quantifying the level of RNA of the bitter taste receptor. In a specific embodiment, the methods described herein for identifying cells having a desired RNA expression profile, wherein the methods comprise: (a) introducing into a cell (e.g., a eukaryotic cell) a plurality of signaling probes each capable of detecting a plurality of RNAs of interest; and (b) quantifying the level of RNA detected by the plurality of signaling probes. The desired gene expression profile can be determined by comparison to a reference population. In particular embodiments, the plurality of RNAs of interest may comprise any combination of RNAs: consisting of SEQ ID NO: 50-102.

In particular embodiments, such methods further comprise the step of comparing the quantified RNA level of the cell to the RNA level in a reference cell, respectively. In particular embodiments, the plurality of signaling probes comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 500, 600, 700, 800, 900, or at least 1000 signaling probes. In certain embodiments, the RNA of interest is translated. In other embodiments, the RNA of interest is not translated. In a specific embodiment, the RNA of interest is encoded by a bitter taste receptor subunit gene. In particular embodiments, the isolated cells express one or more recombinant RNAs of interest.

In particular embodiments, the isolated cells identified and/or selected by the methods described herein have not been genetically engineered (e.g., express one or more transgenes non-recombinantly). In other embodiments, the isolated cells identified and/or selected by the methods described herein have typically been genetically engineered (e.g., recombinantly express one or more transgenes). In particular embodiments, such cells are somatic cells or differentiated cells.

In other embodiments, the cell comprises a desired gene expression profile. In certain embodiments, the desired gene expression profile may be achieved by genetic engineering or by increasing genetic variability. In particular embodiments, the desired gene expression profile can be determined based on a comparison to the gene expression profile of a reference population.

In certain embodiments, the methods described herein are used to identify and/or select cells that express an RNA of interest (e.g., an RNA of a bitter taste receptor) at a higher level than the average heterogeneous cell population (e.g., an unsorted cell line population, such as an unsorted 293T cell population). In certain embodiments, the methods described herein are used to identify and/or select cells that express an RNA of interest (e.g., an RNA of a bitter taste receptor) at a level that is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% higher than the average heterogeneous cell population (e.g., an unsorted cell line population, such as an unsorted 293T cell population). In certain embodiments, the methods described herein are used to identify and/or select cells that express an RNA of interest (e.g., an RNA of a bitter taste receptor) at a level that is at least 1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than the average xenogeneic cell population (e.g., an unsorted population of cell lines, such as an unsorted population of 293T cells).

In particular embodiments, the methods described herein are used to identify and/or select cells that express an RNA of interest (e.g., an RNA of a bitter taste receptor) at a lower level than the average heterogeneous cell population. Exemplary heterogeneous cell populations can be cell populations of mixed cell types of different origin, cell populations of cells of one cell type that are genetically heterogeneous, or cell populations of one particular cell line from which isolated cells are obtained using the methods described herein.

In certain embodiments, the host cell is a human cell. In particular embodiments, the cell is a cell derived from a mouse, rat, monkey, dog, cat, pig, sheep, goat, horse, chicken, frog, worm, insect (e.g., fly), fish, shellfish, or cow. In certain embodiments, the cell is a mammalian cell or a eukaryotic cell. In other embodiments, the cell is a human cell. In certain embodiments, the cell is a primary cell. In other embodiments, the cell is a cell clone of a transformed cell or cell line.

Without being bound by theory, in their role as bitter, bitter taste receptors have putatively evolved to detect a wide variety of potentially physiologically active compounds as bitter. Some of these receptors may be more broadly regulated in their binding or interaction with a broader chemical structure than other GPCRs. Thus, testing bitter GPCRs using a library of structurally diverse compounds can be used to identify compounds that interact with or modulate bitter taste receptors. Thereby predicting that the identified compound also modulates the activity of other GPCRs.

In certain more specific embodiments, a subject group of different bitter taste receptors, e.g., cell lines expressing at least 2, 5, 10, 50, or at least 100 different bitter taste receptors, may be used to identify the relevant chemical space for other GPCRs most closely related to such bitter taste receptors. Conversely, for compounds with a lower probability of interacting with a GPCR, the chemical space in which no interaction with bitter taste receptors is found can be enriched.

In certain embodiments, the definition of GPCR and non-GPCR chemical spaces according to such methods can be used to enrich chemical libraries that may hit in the HTS process, depending on whether a GPCR or non-GPCR is used. Similarly, the same information can be used to guide pharmaceutical chemistry attempts, such as compounds in the development of non-GPCR targets that can be optimized to exclude functionality that can underlie unwanted interaction with GPCRs.

Any assay used in the methods of the invention can be performed in a high throughput format.

In certain embodiments, if the protein complex is a bitter taste receptor, the activity of the bitter taste receptor can be measured using a calcium mobilization assay.

Identification of structural commonalities between compounds

Systems and methods for identifying structural commonalities between compounds that modulate protein complexes are provided. In certain embodiments, the protein complex may be a cell-expressed receptor, which is a G protein-coupled receptor. In particular embodiments, the protein complex may be a bitter taste receptor. Compounds that modulate protein complexes can be identified using any of the systems and methods disclosed herein. Such compounds can be identified, for example, by separately contacting a number of candidate compounds with cells that have been engineered to express a protein complex, and determining the effect of each candidate compound on the activity of the protein complex, wherein the effect can be determined using a plurality of quantitative measurements of the effect of each candidate compound on the biological activity of the protein complex. In particular embodiments, a library of candidate compounds can be screened against a protein complex to identify compounds that can modulate the protein complex. The effect of the compound of interest on the protein complex can be represented by an activity profile. Non-limiting examples of such quantitative measurements include those derived from the assays discussed herein. Each compound of interest can be represented by a molecular representation that describes the structure and other properties of the compound. In particular embodiments, the molecular representation of the compound of interest may include a structural descriptor that mathematically describes the structural characteristics of the compound. Structural and other types of descriptors are described below. A structure-activity relationship (SAR) model can be used to mathematically relate one or more descriptors of a compound of interest to a biological activity of the compound, as observed from an activity spectrum of the compound.

Systems and methods for identifying structural commonalities between compounds that modulate protein complexes are provided, which may include constructing one or more SAR models of a compound of interest using a molecular representation of the compound and an activity profile thereof, identifying one or more structural features of each compound that correlate with the activity profile of the respective compound based on the SAR models, and identifying at least one of the one or more structural features identified for each compound that is common to the compound. In particular embodiments, a molecular representation of the respective compound and an activity profile of the respective compound can be used to construct a SAR model for each compound of interest. The structure-activity relationship (SAR) model is discussed below.

From the SAR model, one or more structural features of each compound of interest can be identified that correlate with the activity profile of the respective compound. From such features, one or more structural features (structural commonalities) common to all compounds of interest can be identified. In certain embodiments, computer modeling techniques can be used to visualize the three-dimensional atomic structure of a compound of interest. In particular embodiments, computer modeling techniques can be used to visualize structural commonalities of compounds of interest. The three-dimensional structure of a molecule may depend on data from X-ray crystallographic analysis or NMR imaging of the selected molecule. The force field data can be used to generate models and/or structural commonalities of the molecular dynamics of the compound of interest. Examples of molecular modeling programs are CHARMm and QUANTA programs (Accelrys, inc., San Diego, CA).

Molecular representation of a Compound

A molecular representation of a compound of interest may include a plurality of descriptors that describe characteristics and properties of the compound. The descriptors may be topological, structural, physicochemical, and/or spatial type descriptors. A given compound may be represented by a number of descriptors, including the sub-structural components or parts of the compound, the distance of chemical functional groups, spatial, 2D or 3D topology, electrochemical, electrophysiological and quantum mechanical properties. In particular embodiments, the compound of interest may be represented by a structural descriptor. Examples of structural descriptors of a compound may include, but are not limited to, atom type, Molecular Weight (MW), number of rotatable bonds (rotenbond) in the compound, number of hydrogen bond donors (H bond donors), number of hydrogen bond acceptors (H bond acceptors), number of chiral neutrals (R or S), 3D molecular moment, substructure properties, molecular properties, and quantum mechanical properties. Non-limiting examples of topology descriptors include Balaban index (Jx), kappa shape index, flexibility, subgraph count, connectivity, Wiener index, Zagreb index, connectivity index, Hosoya index, and E-state key (E-state key). Non-limiting examples of physicochemical (or thermodynamic) descriptors include logarithm of partition coefficient (AlogP), partition coefficient, atomic-type value (atom-type) (AlogP98), logarithm of octanol/water partition coefficient (LogP), molar refractive index (MR), and molar refractive index (MolRef). Non-limiting examples of spatial descriptors include radius of gyration (radOfgyration), Jurs electrical partial surface area (Jurs) descriptors (Jurs), surface area projection (shadow index), molecular surface area (area), density, molecular volume (Vm), and Principal Moment of Inertia (PMI). Non-limiting examples of electronic descriptors include charge, sum of atomic polarizabilities (Apol), highest occupied molecular orbital energy (HOMO), lowest unoccupied molecular orbital energy (LMMO), and super delocalization (Sr). Descriptions of various descriptors can be found in The website called QSAR descriptors, which are available from The scripts Research Institute (LaJolla CA). Definitions can also be found, for example, in websites facing Cerius2 software (Accelrys, inc., San Diego, CA) or Accelrys QSAR software products (Accelrys, inc., San Diego, CA).

The values of multiple descriptors of a compound of interest can be inferred using a computer program for determining the structure of a molecule. For example, the GRID program (Molecular Discovery ltd., Middlesex, UK) can be used to determine energetically favorable binding sites on compounds of known structure that can be used as descriptor inputs in QSAR assays. Likewise, programs that perform QSAR analysis may also be used to infer the value of a descriptor of a compound of interest, such as the Accelrys QSAR software product (Accelrys, inc., San Diego, CA). In another example, information that can be used as a descriptor of a compound of interest can be obtained from a database of compound structures, such as the Accelrys Chemicals Available for use in a Purchase (CAP) database (Accelrys Inc., San Diego, Calif.).

Structural and activity relationship models

The SAR model can be used to relate descriptors of a compound of interest to the biological activity of the compound on the data. The biological activity of the compound of interest can be provided by the activity profile of the compound. Different combinations of descriptors and algorithms can be used to construct SAR models to build models that can be used to classify other compounds. For example, SAR that predicts structural features of a class of compounds that modulate protein complexes, which can be constructed using a set of training compounds that are highly similar to each other, can be used to classify other compounds that are thought to be similar to the training set.

SAR algorithms can be used to describe the mathematical relationship between the relevant chemical descriptors and the potential for observed biological activity, i.e., activity Y is a function of descriptor X, [ Y ═ f (X) ]. Any algorithm used in the art for data interrogation may be used to construct the SAR model. By way of non-limiting example, programs that may be used to construct the SAR model include programs that may perform general multiple regression (OMR), stepwise regression (SWR), all Possible Subset Regression (PSR), and Partial Least Squares (PLS) regression, as well as Genetic Algorithms (GA). In particular embodiments, the SAR model of the compound may take the form of a linear equation:

A0+(A1M1)+(A2M2)+(A3M3)+...(AnMn)

where the parameters M1 to Mn are different descriptors in the molecular representation of the compound, a0 is a constant, and the coefficients a1 to An are calculated by fitting the changes in the parameters M1 to Mn to the biological activity (e.g. provided by An activity spectrum) of the compound. The SAR model can be fitted using any regression technique within the art. For example, a least squares fit of the independent variables (e.g., employed as descriptors) to the dependent variables (e.g., employed as activity spectra) can be calculated using ordinary multivariate regression. Examples of software that can be used to provide a SAR model, for example by performing quantitative structure-activity relationship analysis (QSAR), include, but are not limited to, Accelrys QSAR software (Accelrys inc., San Diego, CA) and Quasar 5, 6D-QSAR software products (Biographies Laboratory 3R, Basel, Switzerland). Other SAR models are described in Selasie, CD., "storage of Quantitative Structure-Activity relationships", Burger's Medicinal Chemistry and Drug Discovery, 6 th edition, volume 1: drug Discovery, which is incorporated herein by reference in its entirety.

Examples of SAR models that can be constructed for a compound of interest include, but are not limited to, receptor-dependent free-ability field QSAR (FEFF-QSAR), receptor-independent three-dimensional QSAR (3D-QSAR), and receptor-dependent or receptor-independent four-dimensional QSAR (4D-QSAR).

3D-QSAR independent of the receptor. A method that can be used to provide a tool that relates the magnitude of a particular property exhibited by a compound to one or more structural features and/or physical properties of the compound is receptor-independent 3D-QSAR analysis. The receptor geometry may be unknown in the performance of a receptor-independent 3D-QSAR assay. Receptor-independent QSARs can be applied to a range of chemical analogs whose dependent (i.e., bioactive) properties are derived from a set of intramolecular descriptors, based on the assumption that compounds share a common mechanism of action. Regression analysis can be used to fit descriptors of compounds to different measures of activity spectra to perform receptor-independent 3D-QSAR analysis.

Receptor-dependent 3D-QSAR. Another method may use the use of receptor-dependent 3D-QSAR (also known as free energy field QSAR (FEFF-QSAR). for receptor-dependent 3D-QSAR analysis, the receptor geometry is known. the free energy field ligand-receptor binding energy term can be calculated and used as an independent variable for the QSAR scoring function see, e.g., Tokarski and Hopfinger (1997), J.chem.Inf.computer Sci.37: 792-811, which is incorporated herein by reference in its entirety.

Receptor-dependent or receptor-independent 4D-QSAR. The 4D-QSAR model can exploit 3D-QSAR analysis to incorporate conformational and alignment freedom by performing a global averaging of molecular states (fourth dimension) on a set of training compounds. In the 4D-QSAR analysis, it is believed that differences in compound activity can be related to differences in the Boltzmann's mean spatial distribution of molecular shape, in terms of the principle of Interacting Pharmacophores (IPE). A single "active" conformation may be assumed for each compound in a set of training compounds. The 4D-QSAR analysis can be used with additional molecular design applications including receptor-independent 3D-QSAR and FEFF-QSAR models. The 4D-QSAR model is described in Duca and Hopfinger (2001), J Chem InfCompout Sci 41 (5): 1367-87, which are incorporated by reference herein in their entirety.

Instrument, computer and computer program product implementation

The present invention may be implemented as a computer program product comprising a computer program mechanism embedded in a computer readable storage medium. Furthermore, any of the methods of the present invention may be performed in one or more computers or other forms of devices. Examples of devices include, but are not limited to, computers and measurement equipment (e.g., assay readers or scanners). Furthermore, any of the methods of the present invention can be implemented in one or more computer program products. Some embodiments of the invention provide a computer program product encoding any or all of the methods disclosed in the application. Such methods may be stored on a CD-ROM, DVD, magnetic disk storage product, or any other computer readable or program storage product. Such computer-readable storage media tend to be tangible physical objects (as opposed to carrier waves). Such methods may also be implanted in permanent memory such as ROM, one or more programmable chips, or one or more Application Specific Integrated Circuits (ASICs). Such persistent storage may be located in a server, 802.11 access point, 802.11 wireless connection/station, repeater, router, mobile phone, or other electronic device. Such methods embedded in a computer program product may also be distributed electronically, via the internet, or otherwise, by transmitting a computer data signal (in which the software module is embedded) either digitally or over a carrier wave (it being clear that such use of the carrier wave is for distribution and not storage).

Some embodiments of the invention provide computer program products. These program modules may be stored on a CD-ROM, DVD, magnetic disk storage product, or any other computer readable data or program storage product. Program modules may also be embedded in permanent memory, e.g., ROM, one or more programmable chips, one or more Application Specific Integrated Circuits (ASICs). Such persistent storage may be located in a server, 802.11 access point, 802.11 wireless connection/station, repeater, router, mobile phone or other electronic device. The software modules in the computer program product may also be distributed electronically, via the internet or otherwise, through transmission of a computer data signal (in which the software modules are embedded) either digitally or on a carrier wave.

In particular embodiments, the computer program provides for outputting the results of the methods to a user, a user interface device, a computer readable storage medium, a monitor, a local computer, or a computer that is part of a network. Such computer storage media tend to be tangible physical objects (as opposed to carriers).

These and other embodiments of the invention can be further illustrated in the following non-limiting examples.

Examples

Example 1 Generation of Stable GABAA expressing cell lines

Generation of expression vectors

Plasmid expression vectors allowing for streamlined cloning were generated based on pCMV-script (stratagene) and contain a variety of desired components for transcription and translation of the gene of interest, including CMV and SV40 eukaryotic promoters; SV40 and HSV-TK polyadenylation sequences; a multiple cloning site; a Kozak sequence and a neomycin/kanamycin resistance cassette.

Step 1: transfection

We transfected 293T and CHO cells. This example focuses on CHO cells co-transfected with 3 separate plasmids in the following combinations: α 1 β 3 γ 2s (. alpha.1), α 2 β 3 γ 2s (. alpha.2), α 3 β 3 γ 2s (. alpha.3) and α 5 β 3 γ 2s (. alpha.5), one plasmid encodes the human GABA α subunit (SEQ ID NOS: 1-4), one plasmid encodes the human GABA β 3 subunit (SEQ ID NO: 5) and the other plasmid encodes the human GABA γ 2 subunit (SEQ ID NO: 6). As will be appreciated by one of ordinary skill in the art, any agent suitable for use with the selected host cell can be used to introduce nucleic acids (e.g., plasmids, oligonucleotides, labeled oligonucleotides) into the host cell with appropriate optimization. Examples of agents that can be used to introduce nucleic acids into a host cell include, but are not limited to, Lipofectamine2000, Oligofectamine, TFX agents, Fugene 6, DOTAP/DOPE, Metafectine, or Fecturin.

Although drug selection is optional in the methods of the invention, we include a drug resistance marker/plasmid. The sequence is under the control of the CMV promoter. The untranslated sequence encoding the tag for detection by the signaling probe is also present along with the sequence encoding the drug resistance marker. The target sequences utilized were GABA target 1(SEQ ID NO: 7), GABA target 2(SEQ ID NO: 8) and GABA target 3(SEQ ID NO: 9). In these examples, the vector comprising the GABA α subunit gene comprises GABA target 1, the vector comprising the GABA β subunit gene comprises GABA target 2, and the vector comprising the GABA γ subunit gene comprises GABA target 3.

Step 2: selection step

Transfected cells were cultured in HAMF12-FBS for 2 days, followed by antibiotic-containing HAMF12-FBS for 14 days. The antibiotic-containing period had the following antibiotics added to the medium: puromycin (3.5. mu.g/ml), hygromycin (150. mu.g/ml) and G418/neomycin (300. mu.g/ml).

And step 3: cell passage

After antibiotic selection, and prior to introduction of the fluorescent probe, cells were passaged 6 to 18 times in the absence of antibiotic to allow time for unstable expression to decline over the selected time period.

And 4, step 4: contacting the cell with a fluorescent probe

Cells were harvested and transfected with GABA signaling probe (SEQ ID NOS: 10-12). As will be appreciated by one of ordinary skill in the art, any agent suitable for use with the selected host cell can be used to introduce nucleic acids (e.g., plasmids, oligonucleotides, labeled oligonucleotides) into the host cell with appropriate optimization. Examples of reagents that can be used to introduce nucleic acids into a host cell include, but are not limited to, Lipofectamine 2000, Oligofectamine, TFX reagens, Fugene 6, DOTAP/DOPE, Metafectine, or F ecturin.

GABA signaling probe 1 binds to GABA target 1, GABA signaling probe 2 binds to GABA target 2 and GABA signaling probe 3 binds to GABA target 3. Cells were then harvested for analysis and sorted using a fluorescence activated cell sorter (Beckman Coulter, Miami, FL) (below).

Target sequence detection by a signaling probe

GABA target 1

5'-GTTCTTAAGGCACAGGAACTGGGAC-3' (SEQ ID NO: 7) (alpha subunit)

GABA target 2

5'-GAAGTTAACCCTGTCGTTCTGCGAC-3' (SEQ ID NO: 8) (beta subunit)

GABA target 3

5'-GTTCTATAGGGTCTGCTTGTCGCTC-3' (SEQ ID NO: 9) (gamma subunit)

Signal conduction probe

As a 100 μ M stock solution supply

GABA signaling probe 1-binding (GABA target 1)

5 '-Cy 5 GCCAGTCCCAGTTTCCTGTGTGCCTTAAGACCTCGC BHQ3 quenching-3' (SEQ ID NO: 10)

GABA signaling probe 2-binding (GABA target 2)

5 '-Cy5.5GCGAGTCGCAGAACGACAGGGTTAACTTCCTCGCBHQ3 quenching-3' (SEQ ID NO: 11)

Similar probes of quasardye (biosearch) with similar spectral properties as Cy5 were used in specific experiments. It should also be noted that in some cases 5-MedC and 2-aminodA mixmer probes are used instead of DNA probes. It should be noted that BHQ3 can be replaced by BHQ2 or gold particles in Probe 1 or Probe 2.

GABA signaling probe 3-binding (GABA target 3)

5 '-Fam GCGAGAGCGACAAGCAGACCTATAGAACCTCGCCBHQ 1 quench-3' (SEQ ID NO: 12)

It should be noted that BHQ1 can be replaced by BHQ2 or Dabcyl in Probe 3.

And 5: isolation of Positive cells

Cells were dissociated and collected for analysis and sorted using a fluorescence activated cell sorter (Beckman Coulter, Miami, FL). Standard assay methods are used to gate cells that fluoresce above background and isolate individual cells that fall within the gate into a barcoded 96-well plate. The gating levels are as follows: and (3) gating level: coincidence gate (coherent gate) > unimodal gate (singlets gate) > valve (live gate) > sorting gate (Sort gate). With this gating strategy, 0.04-0.4% of the top of the triple positive cells were marked for sorting into barcoded 96-well plates.

Step 6: additional cycles of Steps 1-5 and/or 3-5

Repeating steps 1-5 and/or 3-5 to obtain a larger number of cells. 2 independent cycles of steps 1-5 are completed and for each of these cycles, at least 3 internal cycles of steps 3-5 are performed for the aggregation of the independent cycles.

And 7: estimation of the Long Rate of cell populations

The plates were transferred to a Hamilton Microlabstar automated liquid handler. Cells were grown on 2-3 day conditions in medium: fresh growth medium (Ham's F12/10% FBS) in a 1: 1 mixture was incubated for 5-7 days supplemented with 100 units penicillin/ml plus 0.1mg/ml streptomycin and then dispersed by trypsinization with 0.25% trypsin to minimize clotting and transferred to new 96 well plates. After clone dispersion, plates were imaged to determine well confluence (Genetix). Each plate is focused for reliable image acquisition across the plate. Not relying on reported confluencies of more than 70%. Confluency measurements were obtained at every 3 days over 9 days (between days 1 and 10 after dispersion) and used to calculate growth rate.

And 8: boxed cell population estimation from growth rate

The cell frames were pooled (grouped independently and plated as contemporaneous clusters) according to growth rates between 10-11 days after the dispersion step in step 7. Frames are collected independently and spread out on individual 96-well plates for downstream processing, and there may be more than one target plate/specific frame. The boxes were calculated by considering the spread in growth rate and classifying ranges covering a high percentage of the total number of cell populations as homogeneous. Depending on the sorting iteration (see step 5), 5-6 growth boxes with 1-4 day separation were used. Each box thus corresponds to a growth rate or population doubling time difference between 12-14.4 hours, depending on the iteration.

And step 9: repeated coating to speed parallel processing and provide stringent QC

The plates were subjected to standard and fixed conditions (humidified 37 ℃, 5% CO)₂V/95% air) in Ham's F12 medium/10% FBS without antibiotics. The plates of cells were separated to generate 4 sets (a set consisting of all plates for all growth frames-these steps ensure 4 replicates of the starting set) of target plates. Up to 2 target plate groups were submitted for cryopreservation (see below), and the remaining groups were scaled up and further replated for passage and for functional assays. Different and independent tissue culture reagents, incubators, personnel and carbon dioxide sources were used for each plate set performed independently. By quality control Steps to ensure proper production and quality of all tissue culture reagents; each component added to each bottle of media prepared for use was added by one designated person in one designated fume hood, where only that reagent was present, while the second designated person was supervising to avoid errors. Conditions for liquid handling are set to eliminate cross-contamination across the wells. Fresh tips were used for all steps, or a stringent tip washing protocol was used. Liquid handling conditions were set for accurate volume transfer, efficient cell treatment, wash cycles, pipetting speed and positioning, number of pipetting cycles for cell dispersion, and relative position of tip to plate.

Step 10: early passage stock of frozen cell populations

At least 2 sets of plates were frozen at-70 to-80 ℃. Plates in each group were first allowed to reach 70 to 100% confluence. The medium was aspirated and 90% FBS and 10% DMSO were added. The plates were sealed using Parafilm and then surrounded with 1 to 5cm of foam, respectively, and placed into a-80 ℃ freezer.

Step 11: methods and conditions for an initial conversion step to produce VSF

The remaining plate sets were maintained as described in step 9 (above). All cells were performed separately using automated liquid processing steps including media removal, cell washing, trypsin addition and incubation, quenching and cell dispersion steps.

Step 12: normalization method to correct for any remaining variability in growth rate

The consistency and standardization of cells and culture conditions for all cell populations were controlled. Any differences across the plate due to slight differences in growth rates can be controlled by periodic normalization of the number of cells across the plate.

Step 13: characterization of cell populations

Cells were maintained in cell culture for 6-8 weeks to allow for their evolution in vitro under these conditions. During this time course, we observed size, morphology, fragility, response to trypsin digestion or dissociation, circularity/mean circularity after dissociation, percent viability, tendency towards micro-confluence, or other aspects of cell maintenance such as attachment to the culture plate surface.

Step 14: assessment of potential functionality of cell populations under VSF conditions

The cell population is tested using functional criteria. Membrane potential assay kits (Molecular Devices/MDS) were used according to the manufacturer's instructions. Cells were tested in 96 or 384 well plates at various densities and analyzed for responses. Various time points after spreading are used, for example 12 to 48 hours after spreading. Differences in assay response for different coating densities were also tested.

Step 15

Functional responses from experiments performed at low and higher passage numbers were compared to identify cells with the most consistent response over a defined period of 3-9 weeks. Other cellular characteristics that change over time are also noted, including morphology, tendency toward micro-confluence, and time to attach to the culture substrate after plating.

Step 16

Cell populations that meet functional and other criteria are further evaluated to determine those that are most amenable to the generation of viable, stable and functional cell lines. The selected cell population is expanded in larger tissue culture dishes and the characterization steps described above are continued or repeated under these conditions. In this regard, additional standardization steps were introduced for consistent and reliable passaging. These include different plated cell densities, passage times, culture dish size/form and coating, fluidics optimization, cell dissociation optimization (type, volume used and length of time), and washing steps. The assay Z' score was most stable when the assay was performed every few days over the course of 4 weeks in culture.

In addition, cell viability at each passage was determined. Increasing manual intervention and more closely observing and monitoring the cells. This information is used to help identify and select the final cell line that retains the desired properties. Final and backup cell lines showing consistent growth, proper attachment, and functional response were selected.

And step 17: establishment of cell banks

Low passage freezing plates (see above) corresponding to the final and backup cell lines were thawed at 37 ℃, washed 2 times with Ham's F12/10% FBS, and humidified at 37 ℃/5% CO₂Incubation under conditions. The cells were subsequently expanded for a period of 2-3 weeks. Cell banks were established for each final and backup cell line, each cell line bank consisting of 25 vials each with 10,000,000 cells.

Step 18

At least one vial from a cell bank is used to thaw and expand in culture. The resulting cells were tested to confirm that they met the same characteristics as they were originally selected for.

Example 2 GABA _A Validation of cell lines in response to GABA ligands

Evaluation of expression of GABA_ACHO cell lines of (a 1 ss 3 y 2s (. beta.11), (a 2 ss 03 y 2s (. alpha.2), (a 3 ss 3 y 2s (. alpha.3), and a 5 ss 3 y 2s (. alpha.5)) have endogenous GABA_AThe response of the ligand. Using the following protocol, GABA was measured in response to GABA_AMembrane potential, evaluating the interaction of cell lines with GABA.

24 hours prior to assay, cells were plated in growth medium (Ham's F-12 medium plus FBS and glutamine) at 10-25,000 cells/well in 384-well plates. The medium was removed, followed by addition of membrane potential dye diluted in loading buffer (137mM NaCl, 5mM KCl, 1.25mM CaCl, 25mM HEPES, 10mM glucose), incubation for 1 hour, followed by loading the plates into high throughput fluorescent plate reads On a machine (Hamamastu FDSS). GABA ligands were assayed in MP assay buffer (137mM NaCl, 5mM potassium gluconate, 1.25mM CaCl)₂25mM HEPES, 10mM glucose) to the desired concentration (when required, serial dilutions of GABA were generated, concentrations used: 3nM, 10nM, 30nM, 100nM, 300nM, 1 μ M, 3 μ M, 10 μ M), and added to each well. The plate was read for 90 seconds.

Table 6 (below) demonstrates the respective responses of the cell lines generated to GABA ligands. These results indicate GABA production in response to endogenous ligands as expected_ACell lines, physiologically relevant for use in high throughput screening assays. In addition, replicating a well yields a precise EC from well to well₅₀Value, indicating GABA_AHigh reproducibility of cell lines. The Z' values generated using membrane potential measurements were α 1 β 3 γ 2s 0.58, α 2 β 3 γ 2s 0.67, α 3 β 3 γ 2s 0.69, and α 5 β 3 γ 2s 0.62.

Example 3 use of known GABA _A Modulators additional validation of GABA _A Cell lines

GABA was verified by the method described in example 2 using serial dilutions in assay buffer of bicuculline (known antagonist) at 30 μ M, 10 μ M, 3 μ M, 1 μ M, 300nM, 100nM, 30nM_ACell lines and membrane potential assays.

EC found in GABA ₅₀Dicentrine with all 4 GABA in the presence of concentration_ACell line interactions. These results indicate a response to GABA as expected_AThe production of this known modulator of_ACell lines, physiologically and pharmacologically relevant for use in high throughput screening assays.

Example 4 native GABA Using Membrane potential assay _A Functional characterization of expression of GABA _A Cells Is a system

Evaluation of expression of GABA_AInteraction of CHO cell lines (. alpha.1. beta.3. gamma.2 s (. beta.11), (. alpha.2. beta.03. gamma.2 s (. alpha.2), (. alpha.3. beta.3. gamma.2 s (. alpha.3), and. alpha.5. beta.3. gamma.2 s (. alpha.5)) with 1280 Compounds from LOPAC 1280(library of pharmaceutical Active Compounds) (Sigma-RBI product number L01280.) the LOPAC 1280 library contains high purity, small organic ligands with well documented pharmacological activities_AAnd evaluating the interaction of the cell line with the test compound.

24 hours prior to assay, cells were plated in growth medium (Ham's F-12 medium plus FBS and glutamine) at 10-25,000 cells/well in 384-well plates. The medium was removed and then added to a loading buffer (137mM NaCl, 5mM KCl, 1.25mM CaCl) ₂25mM HEPES, 10mM glucose). Incubation was 1 hour, and the plates were subsequently loaded onto a high energy fluorescent plate reader (Hamamastu FDSS). Test compounds were placed in MP assay buffer (137mM NaCl, 5mM potassium gluconate, 1.25mM CaCl₂25mM HEPES, 10mM glucose) to the desired concentration (when needed, serial dilutions of each test compound were made, concentrations used: 3nM, 10nM, 30nM, 100nM, 300nM, 1 μ M, 3 μ M, 10 μ M), and added to each well. The plate was read for 90 seconds.

Results

Measurement of GABA produced for each compound_AActivity of cell lines and compounds showing similar or greater activity to GABA (endogenous ligand) scored as positive hits. Of the 1280 compounds screened, 34 activated at least one cell line (i.e., either α 1, α 2, α 3 and α 5), and if not better than GABA. 17 of these compounds and GABA produced_ACellular interactions were confirmed in the dose response studies described below. Modulators, partial agonists and low potency compounds requiring the presence of GABA are not included in the list.

Screening assays to identify each GABA in LOPAC libraries_AAgonist(s): GABA (endogenous ligand), propofol, isonorbetuline hydrochloride, muscimol hydrobromide, piperidine-4-sulfonic acid, 3- α, 21-dihydroxy-5- α -pregnan-20-one (neurosteroid), 5- α -pregnan-3 α -ol-11, 20-dione (neurosteroid), 5- α -pregnan-3 α -ol-20-one (neurosteroid), and tracarbazolyl. The results indicate that GABA is produced _ACell lines respond in a physiologically relevant manner (e.g., they respond to agonists of endogenous receptors). Determination of EC for these 8 agonists₅₀Values and are included in table 6 (below).

The screening assay also identified compounds that are not described as GABA agonists in the LOPAC library but are known to have properties that are comparable to GABA_A4 compounds of relevant other activities, which were noted to be: etazolate (phosphodiesterase inhibitor), androsterone (steroid hormone), chlormezanone (muscle relaxant), and ivermectin (antiparasitic known to affect chloride channels). Determination of EC for these 4 Compounds₅₀Values, and are summarized in table 6 (below).

The screening assay further identifies the hitherto unknown and GABA in the LOPAC library_A4 compounds that interact. These novel compounds include: dipyridamole (dipyridamole) (adenosine deaminase inhibitor), niclosamide (antiparasitic) tyrphosinA9(PDGFR inhibitor) and I-Ome-Tyrphosin AG 538 (RTK IGF inhibitor). Determination of EC for these 4 Compounds₅₀Values, and are summarized in table 6 (below).

The results of the screening assay are summarized in table 6:

example 5 native GABA Using electrophysiological measurements _A Functional characterization of GABA _A -CHO Cells

The following potential clamp protocol was used: the membrane potential was clamped to a holding potential of-60 mV. Excitation current was applied by 2 seconds of increasing concentration of GABA (0.10-100. mu.M) with intermediate washes with buffer.

GABA for alpha 2, alpha 3 and alpha 5_ACell line response 100 μ M GABA, and α 1GABA_AWhole cell receptor current tracking of cell lines in response to increasing concentrations of GABA (0.10-100 μ M in logarithmic increments) demonstrated that GABA is in addition to the high throughput screening assay described above_ACell lines can also be used in conventional electrophysiological assays. These electrophysiological measurements, along with the membrane potential measurements of example 2, confirm the GABA produced herein_ACell line physiological and pharmacological relevance. Electrophysiology is well known as detection of GABA_AA reliable method for the modulation of receptors. Our data indicate that the cell lines of the invention can produce similar reliable results using membrane potential assays. The cell lines of the prior art are not reliable enough or sensitive enough to make efficient use of such membrane potential assays, which are cheaper and faster than electrophysiology. Thus, the cell lines of the invention allow screening on a much larger scale than is available using electrophysiology (10,000 assays/day using membrane potential compared to less than 100 assays/day using electrophysiology).

Example 6 use of halide sensitive meYFP on native GABA _A Functional intracellular Characterization of the read measurements

Evaluation of the expression of GABA according to the invention Using the following protocol for intracellular readout assay _A(α1β3γ2s(A1)、(α2β3γ2s(A 2)、(α3β3γ2s(A 3)、And α 5 β 3 γ 2s (A5) to a test compound.

24 hours prior to assay, cells were plated in growth medium (Ham's F-12 culture plus FBS and glutamine) at 10-25,000 cells/well in 384-well plates. The medium was removed and then loading buffer (135mM NaCl, 5mM KCl, 2mM CaCl) was added₂、1mMMgCl₂10mM HEPES, 10mM glucose) and incubated for 1 hour. The assay plates were then loaded onto FDSS (Hamamatsu corporation). Test compounds (e.g., GABA ligands) were placed in assay buffer (150mM NaCl, 5mM KCl, 1.25mM CaCl)₂、1mM MgCl₂25mM HEPES, 10mM glucose) to the desired concentration (when required, to produce serial dilutions of each test compound, effective concentration used: 3nM, 10nM, 30nM, 100nM, 300nM, 1 μ M, 3 μ M, 10 μ M), and added to each well. The plate was read for 90 seconds.

GABA in response to increasing concentrations of GABA ligands_A-meYFP-CHO cells showed increased quenching of the meYFP signal. This quenching can be used to calculate a dose response curve for GABA activation. The GABA dose response curve generated by the intracellular readout assay is similar to the curve generated by the membrane potential blue assay described in example 3. These data demonstrate that the cells of the invention can be used to measure GABA in an intracellular read assay _AThe regulator of (1).

Example 7 Generation of Stable GC-C expressing cell lines

293T cells were transfected with a plasmid encoding the human GC-C gene (SEQ ID NO: 15) using standard techniques. (examples of reagents that can be used to introduce a nucleic acid into a host cell include, but are not limited to, LIPOFECTAMINE^TM、LIPOFECTAMINE^TM 2000、OLIGOFECTAMINE^TM、TFX^TMA reagent,DOTAP/DOPE, metaflecine or FECTURIN^TM。)

Although drug selection is optional in the methods of the invention, we include a drug resistance marker in the plasmid encoding the human GC-C gene. The GC-C sequence is under the control of the CMV promoter. The untranslated sequence encoding the tag for detection by the signaling probe is also present along with the sequence encoding the drug resistance marker. The target sequence utilized was GABA target sequence 1(SEQ ID NO: 13). In this example, the vector containing the GC-C gene contains the GC-C target sequence 1.

Transfected cells were cultured in DMEM-FBS for 2 days, then in DMEM-FBS containing 500 μ g/ml hygromycin for 10 days, then in DMEM-FBS for the remainder of the time, in DMEM/10% FBS for a total of 4 to 5 weeks (depending on which individual separation) before addition of the signaling probe.

After enrichment on antibiotics, cells were passaged 8-10 times in the absence of antibiotic selection to allow for time for unstable expression to decline over a selected period of time.

Cells were harvested and transfected with GC-C signaling probe 1(SEQ ID NO: 14) using standard techniques. (examples of reagents that can be used to introduce a nucleic acid into a host cell include, but are not limited to, LIPOFECTAMINE^TM、LIPOFECTAMINE^TM2000、OLIGOFECTAMINE^TM、TFX^TMA reagent,DOTAP/DOPE, metaflecine or FECTURIN^TM. ) Cells were then dissociated and collected for analysis and sorted using a fluorescence activated cell sorter (Beckman Coulter, Miami, FL).

GC-C target sequence 1 detected by GC-C signaling probe 1

5′-GTTCTTAAGGCACAGGAACTGGGAC-3′(SEQ ID NO：13)

GC-C signaling probe 1

(supplied as 100. mu.M stock solution)

5′-Cy5 GCCAGTCCCAGTTCCTGTGCCTTAAGAACCTCGCBHQ2-3′(SEQ ID NO：14)

Furthermore, those with similar spectral properties to Cy5 were used in specific experiments(BioSearch) similar probes. In some experiments, 5-MedC and 2-aminodAmixmer probes were used instead of DNA probes.

Cells were dissociated and collected for analysis and sorted using a fluorescence activated cell sorter (Beckman Coulter, Miami, FL). Standard analytical methods were used to gate cells that fluoresced above background and individual cells that were included in the gate were isolated into barcoded 96-well plates. In cell FAM compared to Cy 5: the following gating levels were used in 0.3% live cells:

coincidence gate → unimodal gate → trap door → sorting gate.

The above procedure was repeated to obtain a larger number of cells. All 2 rounds of the above steps were performed. In addition, for one of the independent transfection rounds, the sequence of steps of cell passaging, contacting with signaling probes, and isolating positive cells by fluorescence-activated cell sorter was performed a total of 2 times.

Transfer plates to MICROLAB STAR^TM(Hamilton Robotics). Cells were incubated for 9 days in 100 μ l of a 1: 1 mixture of fresh complete growth medium and 2 days conditioned growth medium supplemented with 100U of penicillin and 0.1mg/ml daptomycin, dispersed 2 times by trypsinization to minimize clotting, and transferred to new 96-well plates. The plates were imaged to determine well confluence (Genetix). Each plate is focused for reliable image acquisition across the plate. Not relying on reported confluencies of more than 70%. Confluent measurements were obtained at 3 consecutive days and used to calculate growth rate.

The cells were boxed (grouped independently and plated as a contemporaneous group) according to the growth rate 3 days after the dispersion step. 4 growth frames each separated into a respective 96-well plate; some growth frames produce more than one 96-well plate. The boxes were calculated by considering the spread in growth rate and classifying ranges covering a high percentage of the total number of cell populations as homogeneous. Boxes were calculated to capture the 12 hour difference in growth rate.

Cells may have doubling times of less than 1 day to over 2 weeks. For processing the most dispersed clones, which at the same time can be reasonably boxed according to growth rate, it is preferred to use 3-9 boxes with a doubling time of 0.25-0.7 days per box. It will be appreciated by those of ordinary skill in the art that the closeness of the boxes and the number of boxes can be adjusted for a particular situation, and that if the cells are synchronized according to their cell cycle, the closeness and number of boxes can be further adjusted.

The plates were subjected to standard and fixed conditions (DMEM/FBS, 37 ℃, 5% CO)₂) Incubate without antibiotics. The plates of cells were split to generate 5 sets of 96-well plates (3 for freezing, 1 for assay and 1 for passaging). Different and independent tissue culture reagents, incubators, personnel and carbon dioxide sources were used downstream of the working stream for each plate set. Quality control steps were taken to ensure proper production and quality of all tissue culture reagents: each component added to each bottle of media prepared for use was added by one designated person in one designated fume hood, where only that reagent was present, while the second designated person was supervising to avoid errors. Conditions for liquid handling are set to eliminate cross-contamination between wells. Fresh tips were used for all steps, or a stringent tip washing protocol was used. Liquid handling conditions were set for accurate volume transfer, efficient cell treatment, wash cycles, pipetting speed and positioning, number of pipetting cycles for cell dispersion, and relative position of tip to plate.

Group 1 plates were frozen at-70 to-80 ℃. Plates in the set were first allowed to reach 70-100% confluence. The medium was aspirated and 90% FBS and 10% DMSO were added. The plates were individually sealed with Parafilm, surrounded by 1-5cm of foam, and placed in a refrigerator.

The remaining 2 sets of plates were maintained under standard and fixed conditions as described above. All cells were performed separately using automated liquid processing steps including media removal, cell washing, trypsin addition and incubation, quenching and cell dispersion steps.

The consistency and standardization of cells and culture conditions for all cell populations were controlled. Differences across the plate due to slight differences in growth rate were controlled by normalization of the number of cells across the plate, and occurred 3 passages after rearrangement. Cell populations that are outliers are detected and eliminated.

Cells were maintained for 3-6 weeks to allow for their evolution in vitro under these conditions. During this time course, size, morphology, tendency towards micro-confluence, fragility, response to and average circularity after trypsinization, or other aspects of cell maintenance such as adhesion to the surface of the culture plate and resistance to ejection after fluid addition were observed.

The cell population is tested using functional criteria. According to the manufacturer's instructions: (http:// www.assaydesigns.com/objects/catalog// product/extract/900-014. pdf) Using the Direct Cyclic GMP EnzymeImmunoassay Kit (catalog 900-014; assayDesigns, Inc.). Cells were tested in 96 or 384 well plates at 4 different densities and analyzed for responses. The following conditions were used for the GC-C expressing cell lines of the present invention:

cloning and screening: resolution of 96-well plates confluent 48 hours prior to assay at 1: 2 and 1: 3 with 30 minutes guanylin treatment.

Dose response study: density of 20,000, 40,000, 60,000, 80,000, 120,000 and 160,000 per well, 30 minutes guanylin treatment (see example 8).

Study Z': densities of 160,000 and 200,000/well, 30 minutes guanylin treatment (see example 10).

Functional responses from experiments performed at low and higher passage numbers were compared to identify cells with the most consistent response over a defined period of 4-10 weeks. Other cellular features that change over time are also noted.

Cell populations that meet functional and other criteria are further evaluated to determine those that are most amenable to the generation of viable, stable and functional cell lines. The selected cell population is expanded in a larger tissue culture vessel and the characterization steps described above are continued or repeated under these conditions. In this regard, additional normalization steps are introduced, such as different cell densities; the time of spreading, the length of cell culture passage; cell culture dish form and coating; fluidics optimization, including velocity and shear; passage time; and a washing step for consistent and reliable passage. In addition, cell viability at each passage was determined. Increasing manual intervention and more closely observing and monitoring the cells. This information is used to help identify and select the final cell line that retains the desired properties. Such final and backup cell lines (20 clones in total) were selected, which when generated according to this process and under these conditions, showed suitable adhesion/viscosity and growth rate and even plating (lack of micro-confluence).

Starting frozen stocks of 3 vials each of the selected 20 clones were generated by amplifying non-frozen populations from the rearranged 96-well plates in DMEM/10% FBS/HEPES/L-Glu via 24-well, 6-well and 10cm dishes. The low passage frozen stocks corresponding to the final cell line and the backup cell line were thawed at 37 ℃, washed 2 times with DMEM containing FBS, and incubated in the same manner. The cells were subsequently expanded for a period of 2-4 weeks. 2 final clones were selected.

At least one vial from an initially frozen one of the clones was thawed and expanded in culture. The resulting cells were tested to confirm that they met the same characteristics according to which they were originally selected. A cell bank consisting of 20 to over 100 vials for each cell line can be established.

The following steps may also be performed to confirm that the cell line is viable, stable and functional: thawing at least one vial from a cell bank and expanding in culture; the resulting cells are tested to determine whether they meet the same characteristics according to which they were originally selected.

Example 8 characterization of cell lines for GC-C Natural function

Competitive ELISA for detection of cGMP was used to characterize native GC-C function in the GC-C expressing cell lines generated. GC-C expressing cells were maintained under standard cell culture conditions in Darbeke Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, glutamine and HEPES and grown in T175cm flasks. For ELISA, cells were plated into coated 96-well plates (poly D-lysine).

Cell processing and cell lysis protocols

Cells were washed 2 times with serum-free medium and incubated with 1mM IBMX for 30 minutes. The desired activator (i.e., guanylin, 0.001-40 μ M) is then added to the cells and incubated for 30-40 minutes. The supernatant was removed and the cells were washed with TBS buffer. Cells were lysed with 0.1N HCl. This was followed by lysis with 0.1N HCI and freeze/thaw cycles at-20 deg.C/room temperature. The thawed lysates (samples were spun in EppendorF tubes at 10,000 rpm) were centrifuged to clot the cell debris. The clarified supernatant lysate was then transferred to an ELISA plate.

ELISA protocol

All following steps were performed at room temperature unless otherwise stated. ELISA plates were coated overnight with anti-IgG antibody in coating buffer (sodium carbonate/sodium bicarbonate buffer, final 0.1M, pH 9.6) at 4 ℃. The plates were then washed with wash buffer (TBS-Tween20, 0.05%) followed by blocking reagent addition. Incubation with blocking reagent at 37 ℃ for 1 hour, followed by washing bufferThe plates were washed with liquid. Rabbit anti-cGMP polyclonal antibody (Chemicon) was then added, followed by incubation for 1 hour and subsequent washing with wash buffer. Cell lysates were then added and incubated for 1 hour before subsequent addition of cGMP-biotin conjugate (1 and 10nM 8-biotin-AET-cGMP (biolog)). The plates were incubated for 2 hours and then washed with wash buffer. Streptavidin basic phosphate was then added and incubated for 1 hour, followed by washing with wash buffer. The plates were incubated with PNPP substrate (Sigma) for at least 1 hour (preferably 2-5 hours). Then in SAFIRE ^2TMThe absorbance at 405nm was read on a plate reader (Tecan).

The maximum absorbance was seen when no cell lysate was used in the ELISA (control). A decrease in absorbance (corresponding to increased cGMP levels) was observed with cell lysates from the generated GC-C expressing cell lines treated with 100nM guanylin (clone).

The cGMP levels in the resulting GC-C expressing cell lines treated with 100nM guanylin were also compared to that of a control sample of the parental cell line that does not express GC-C (not shown) using the Direct Cyclic GMP Enzyme Immuassay Kit (catalog 900-014; AssayDesigns, Inc.). GC-C expressing cell lines showed a greater decrease in absorbance (corresponding to increased cGMP levels) compared to the parental cells treated with guanylin and untreated.

For guanylin dose response experiments, cells of the resulting GC-C expressing cell lines plated at densities of 20,000, 40,000, 60,000, 80,000, 120,000, and 160,000 cells/well in 96-well plates were challenged with increasing concentrations of guanylin for 30 minutes. Using SAFIRE^2TMPlate reader (Tecan), detecting cellular responses (i.e., absorbance) as a function of changes in cGMP levels (as measured using the Direct Cyclic GMP enzymelmumunomassaykit (catalog 900-014; AssayDesigns, Inc.). data was then plotted as a function of guanylin concentration and analyzed using nonlinear regression analysis using GraphPad Prism 5.0 software, yielding an EC of 1.1nM ₅₀The value is obtained. And previously reported in other cell linesComparison (3.5pmol/ml) (Fore et al, Endocr.140 (4): 1800-. The resulting GC-C expressing cell lines showed higher levels of cGMP (6pmol/ml) when treated with low concentrations of guanylin, indicating the potency of the clones.

EXAMPLE 9 Generation of Z' values for GC-C expressing cell lines

Z' was calculated for the GC-C expressing cell lines generated using a direct competitive ELISA assay. ELISA was performed using the Direct Cyclic GMP Enzyme Immunoassay Kit (catalog 900-014; assay designs, Inc.). Specifically, for the Z' assay, 24 positive control wells (plated at a density of 160,000 or 200,000 cells/well) in a 96-well assay plate were challenged with 40 μ M of a GC-C activation mixture (cocktail) of guanylin and IBMX in DMEM medium for 30 minutes. Considering the volume and surface area of the 96-well assay plate, this amount of guanylin produced a concentration comparable to the 10 μ M used by Forte et al (1999) Endocr.140(4), 1800-. An equal number of wells containing clonal cells in DMEM/IMBX were challenged with vehicle alone (in the absence of activator). Using SAFIRE^2TMThe plate reader (Tecan) monitored the absorbance (corresponding to cGMP levels) in 2 conditions. The mean and standard deviation in 2 conditions were calculated and the mean and standard deviation were calculated using Zhang et al, J Biomol Screen, 4 (2): 67-73(1999)) calculates Z'. The Z' value of the resulting GC-C expressing cell line was determined to be 0.72.

Example 10 short-circuit current measurement

GC-C expressing cell lines (primary or immortalized epithelial cells such as lung, intestine, breast, uterus or kidney) were plated on culture inserts (snapwell, corning Life Sciences) 7-14 days after which Ussing chamber experiments were performed. Cells on culture inserts were rinsed, fixed on a ewings-type instrument (easysmountchchamber System, photoslogic Instruments), and treated with a continuously aerated ringer solution (in O) maintained at 37 ℃₂5% CO in₂pH 7.4) bath, the ringer solution comprising (in mM)Meter) 120NaCl, 25NaHCO₃、3.3KH₂PO₄、0.8K₂HPO₄、1.2CaCl₂、1.2MgCl₂And 10 glucose. The half-chambers (hemichambers) were connected to a multi-channel potential and current clamp (VCC-MC8, Physiologic Instruments). Use of an electrode [ agar bridged (4% in IM KCl) -Ag-AgCl]And the insert voltage was clamped to 0 mV. Transepithelial current, voltage and resistance were measured every 10 seconds for the duration of the experiment. Films with resistances < 200mOhms were discarded. This secondary assay may provide confirmation that in the appropriate cell type (i.e., tightly-connected cells) the introduced GC-C alters CFTR activity and modulates transepithelial current.

Example 11 Generation of Stable CFTR-expressing cell lines

Generation of expression constructs

Plasmid expression vectors allowing for streamlined cloning were generated based on pCMV-script (stratagene) and contain various desired components for transcription and translation of the gene of interest, including: CMV and SV40 eukaryotic promoters; SV40 and HSV-TK polyadenylation sequences; a multiple cloning site; a Kozak sequence and a drug resistance cassette (i.e., puromycin). Ampicillin or neomycin may also be used in place of puromycin. The tag sequence comprising the target sequence 2(SEQ ID NO: 138) was then inserted into the multiple cloning site of the plasmid. The cDNA cassette encoding human CFTR was then subcloned into the multiple cloning site upstream of the tag sequence using Asc1 and Pac1 restriction endonucleases.

Generation of cell lines

Step 1: transfection

CHO cells were transfected with a plasmid encoding human CFTR (SEQ ID NO: 16) using standard techniques. (examples of reagents that can be used to introduce a nucleic acid into a host cell include, but are not limited to, LIPOFECTAMINE^TM 、LIPOFECTAMINE^TM2000、OLIGOFECTAMINE^TM、TFX^TMA reagent,DOTAP/DOPE, metaflecine or FECTURIN^TM。)

Although drug selection is optional for the generation of the cells or cell lines of the invention, we include a drug resistance marker (i.e., puromycin) in the plasmid. The CFTR sequence is under the control of the CMV promoter. Untranslated sequences encoding CFTR target sequences for detection by signaling probes are also present along with sequences encoding drug resistance markers. The target sequence utilized is CFTR target sequence 2(SEQ ID NO: 138), and in this example, the vector containing the CFTR gene contains CFTR target sequence 2(SEQ ID NO: 138).

Step 2: selecting

Transfected cells were grown in Ham's F12-FBS medium (SigmaAldrich, St. Louis, MO) without antibiotics for 2 days, followed by 10 days in Ham's F12-FBS with 12.5. mu.g/ml puromycin. Subsequently, for the rest of the time, cells were transferred to Ham's F12-FBS medium without antibiotics before addition of the signaling probe.

And step 3: cell passage

After enrichment on antibiotics, cells were passaged 5-14 times in the absence of antibiotic selection to allow for time for unstable expression to decline over the selected time period.

And 4, step 4: contacting the cell with a fluorescent probe

Cells were harvested and transfected with CFTR signaling probe 2(SEQ ID NO: 139) using standard techniques. (examples of reagents that can be used to introduce a nucleic acid into a host cell include, but are not limited to, LIPOFECTAMINE^TM、LIPOFECTAMINE^TM2000、OLIGOFE CTAMINE^TM、TFX^TMA reagent,DOTAP/DOPE, metaflecine or FECTURIN^TM. ) CFTR signaling probe 2(SEQ ID NO: 139) binding to CFTR targetSEQ ID NO: 138. Cells were then collected for analysis and sorted using a fluorescence activated cell sorter.

Detection of target sequences by signaling probes

CFTR target sequence 1

5′-GTTCTTAAGGCACAGGAACTGGGAC-3′(SEQ ID NO：17)

CFTR target sequence 2

5′-GAAGTTAACCCTGTCGTTCTGCGAC-3′(SEQ ID NO：138)

Signal conduction probe

CFTR Signaling Probe 1 (supplied as 100. mu.M stock solution)

5′-Cy5GCCAGTCCCAGTTCCTGTGCCTTAAGAACCTCGCBHQ2-3′(SEQ ID NO：18)

CFTR Signaling Probe 2 (supplied as 100. mu.M stock solution)

5′-CY5.5GCGAGTCGCAGAACGACAGGGTTAACTTCCTCGCBHQ2-3′(SEQ ID NO：139)

The BHQ2 in the signaling probe 2 may be replaced by BHQ3 or gold particles. Target sequence 2 and signaling probe 2 can be replaced by target sequence 1(SEQ ID NO: 17) and signaling probe 1(SEQ ID NO: 18), respectively.

The BHQ2 of the signaling probe 1 may be replaced by BHQ3 or gold particles.

Furthermore, those with similar spectral properties as Cy5 were used in specific experiments against target sequence 1(SEQ ID NO: 17)(BioSearch) similar probes. In some experiments, 5-MedC and 2-aminodA mixmer probes were used instead of DNA probes. Non-targeted FAM labeled probes were also used as loading controls.

And 5: isolation of Positive cells

Cells were dissociated and collected for analysis and sorted using a fluorescence activated cell sorter (Beckman Coulter, Miami, FL). Standard analytical methods were used to gate cells that fluoresced above background and individual cells that were included in the gate were isolated into barcoded 96-well plates. FAM was compared to Cy5 in cell according to standard methods within the art: the following gating levels were used in 0.1-0.4% of live cells:

coincidence gate → unimodal gate → trap door → sorting gate.

Step 6: additional cycles of Steps 1-5 and/or 3-5

Repeating steps 1-5 and/or 3-5 to obtain a larger number of cells. 2 rounds of steps 1-5 are performed and for each of these rounds 2 inner loops of steps 3-5 are performed.

And 7: estimation of growth rate of cell population

The plates were transferred to Microlab Star (Hamilton Robotics). Cells were incubated for 9 days in 100. mu.l of a 1: 1 mixture of fresh complete growth medium and 2-3 days conditioned growth medium supplemented with 100 units/ml penicillin and 0.1mg/ml streptomycin. Cells were then dispersed 1 or 2 times by trypsinization to minimize clumping, and then transferred to new 96-well plates. The plates were imaged to determine well confluence (Genetix). Each plate is focused for reliable image acquisition across the plate. Not relying on reported confluencies of more than 70%. Confluency measurements were obtained on consecutive days between 1 and 10 days after dispersion and used to calculate growth rate.

And 8: boxed cell population estimation from growth rate

The cell frames were pooled (grouped independently and plated as contemporaneous groups) according to a growth rate of less than 2 weeks after the dispersion step in step 7. 3 growth frames each separated into a respective 96-well plate; some growth frames produce more than one 96-well plate. Boxes were calculated by considering the spread of growth rate and classifying a high percentage of the total number of cell populations as homogeneous. Boxes were calculated to capture 12-16 hour differences in growth rate.

Cells may have doubling times of less than 1 day to over 2 weeks. For processing the most dispersed clones, which at the same time can be reasonably boxed according to growth rate, it is preferred to use 3-9 boxes with a doubling time of 0.25-0.7 days per box. It will be appreciated by those of ordinary skill in the art that the closeness of the boxes and the number of boxes can be adjusted for a particular situation, and that if the cells are synchronized with respect to their cell cycle, the closeness and number of boxes can be further adjusted.

And step 9: repeated coating to speed parallel processing and provide tight quality control

The plates were placed in standard and fixed conditions (i.e., Ham's F12-FBS medium, 37 ℃/5% CO)₂) Incubate without antibiotics. The plates of cells were split to generate 4 sets of 96-well plates (3 for freezing and 1 for assay and passaging). Different and independent tissue culture reagents, incubators, personnel and carbon dioxide sources were used for each plate set. Quality control steps were taken to ensure proper production and quality of all tissue culture reagents: each component added to each bottle of media prepared for use was added by one designated person in one designated fume hood, where only that reagent was present, while the second designated person was supervising to avoid errors. Conditions for liquid handling are set to eliminate cross-contamination between wells. Fresh tips were used for all steps, or a stringent tip washing protocol was used. Liquid handling conditions were set for accurate volume transfer, efficient cell treatment, wash cycles, pipetting speed and positioning, number of pipetting cycles for cell dispersion, and relative position of tip to plate.

Step 10: early passage stock of frozen cell populations

Groups 3 plates were frozen at-70 to-80 ℃. Plates in the set were first allowed to reach 70-100% confluence. The medium was aspirated and 90% FBS and 10% DMSO were added. The plates were individually sealed with Parafilm, individually surrounded by 1-5cm of foam, and then placed in a-80 ℃ freezer.

Step 11: methods and conditions for an initial transformation step of a viable, stable and functional (VSF) cell line

The remaining plate sets were maintained as described in step 9. All cells were performed separately using automated liquid processing steps including media removal, cell washing, trypsin addition and incubation, quenching and cell dispersion steps.

The consistency and standardization of cells and culture conditions for all cell populations were controlled. Differences across the plate due to slight differences in growth rate were controlled by normalization of the number of cells across the plate, and occurred after each 8 passages of rearrangement. Cell populations that are outliers are detected and eliminated.

Step 13: characterization of cell populations

Cells were maintained in culture for 6-10 weeks after rearrangement to allow for their evolution in vitro under these conditions. During this time course, we observed size, morphology, tendency towards micro-confluence, fragility, response to and mean circularity after trypsinization, or other aspects of cell maintenance such as adhesion to the culture plate surface and resistance to ejection after fluid addition as part of conventional internal quality control to identify robust cells. Such benchmarked cells are then approved for functional assessment.

The cell population is tested using functional criteria. Membrane potential dye kits (Molecular Devices, MDS) were used according to the manufacturer's instructions.

Cells were plated in 384-well plates in various formatsDensity (i.e., 12.5 × 10)³To 20X 10³Cells/well) were tested and responses were analyzed. The time between cell plating and assay reading is tested. Dye concentrations were also tested. Dose response curves and Z' scores were calculated as part of the potential functional assessment.

The following steps (i.e., steps 15-18) may also be performed to select final and backup viable, stable, and functional cell lines.

Step 15:

functional responses from experiments performed at low and higher passage numbers were compared to identify cells with the most consistent response over a defined period of time (e.g., 3-9 weeks). Other cellular features that change over time are also noted.

Step 16:

cell populations that meet functional and other criteria are further evaluated to determine those that are most amenable to the generation of viable, stable and functional cell lines. The selected cell population is expanded in a larger tissue culture vessel and the characterization steps described above are continued or repeated under these conditions. In this regard, additional standardized steps (e.g., different cell densities, time of plating, length of cell culture passage; cell culture dish form and coating; fluidics optimization including speed and shear, passage time, and washing steps) were introduced for consistent and reliable passage.

In addition, cell viability at each passage was determined. Increasing manual intervention and more closely observing and monitoring the cells. This information is used to help identify and select the final cell line that retains the desired properties. The final and backup cell lines were selected to exhibit appropriate adhesion/viscosity, growth rate and even plating (lack of micro-confluency) when generated according to this procedure and under these conditions.

And step 17: establishment of cell banks

The low passage frozen stocks corresponding to the final and backup cell lines were thawed at 37 ℃, washed 2 times with Ham's f12-FBS, and subsequently incubated in Ham's F12-FBS. The cells were subsequently expanded for a period of 2-4 weeks. A cell bank of clones for each final and backup cell line was established with 25 vials for each clone cell cryopreserved.

Step 18:

at least one vial from the cell bank is thawed and expanded in culture. The resulting cells are tested to determine whether they meet the same characteristics from which they were originally selected.

Example 12 characterization of Stable cell lines for Natural CFTR function

We used a high-throughput compatible fluorescent membrane potential assay to characterize native CFTR function in the resulting stable CFTR-expressing cells.

CHO cell lines stably expressing CFTR were maintained under standard cell culture conditions in Ham's F12 medium supplemented with 10% fetal bovine serum and glutamine. Cells were harvested from the stock plates 1 day prior to assay and plated into 384-well assay plates with black, clear bottoms. The assay plate was maintained at 37 ℃ in a cell culture incubator at 5% CO ₂And the next 22-24 hours. The medium was then removed from the assay plates and added in a loading buffer (137mM NaCl, 5mM KCl, 1.25mM CaCl)₂25 mhepes, 10mM glucose) and allowed to incubate at 37 ℃ for 1 hour. The assay plate was then loaded onto a fluorescence plate reader (Hamamatsu FDSS) and added in compound buffer (137mM sodium gluconate, 5mM potassium gluconate, 1.25mM CaCl₂25 mhepes, 10mM glucose) diluted forskolin and IBMX.

Representative data from the fluorescent membrane potential assay showed that the ion currents attributable to functional CFTR were all higher in stable CFTR-expressing CHO cell lines (cell lines 1, M11, J5, E15 and 015) than in control cells lacking CFTR, as indicated by the assay response.

The ion currents attributable to functional CFTR in stable CFTR expressing CHO cell lines (cell lines 1, M11, J5, E15 and O15) were also all higher than in transient CFTR transfected CHO cells. Transient CFTR transfected CHO cells were generated by plating CHO cells on 5-16,000,000/10cm tissue culture dishes and incubating them for 18-20 hours prior to transfection. A transfection complex consisting of lipofectin and plasmid encoding CFTR was added directly to each dish. The cells were then incubated at 37 ℃ in CO ₂Incubate in incubator for 6-12 hours. After incubation, cells were extracted, plated into 384-well assay plates with black, clear bottoms, and their function was determined using the fluorescent membrane potentiometry described above.

For forskolin dose response experiments, stable CFTR expressing cell lines generated, plated at a density of 15,000 cells/well in 384-well plates, were challenged with increasing concentrations of forskolin (a known CFTR agonist). Cellular responses as a function of changes in cellular fluorescence were monitored over time by a fluorescence plate reader (Hamamatsu FDSS). The data was then plotted as a function of forskolin concentration and analyzed using nonlinear regression analysis using GraphPad Prism 5.0 software, yielding an EC of 256nM₅₀The value is obtained. The resulting CFTR-expressing cell lines showed forskolin EC previously reported in other cell lines₅₀EC for forskolin in the range of (250-500nM) (Galietta et al, Am J Physiol Cell physiol.281 (5): C1734-1742(2001))₅₀Value, indicating the efficacy of the clone.

Example 13 determination of Z' values based on CFTR cell-based assays

Z' values for the generated stable CFTR expressing cell lines were calculated using high-throughput compatible fluorescent membrane potentiometry. The fluorescent membrane potential assay protocol was performed essentially according to the protocol in example 12. Specifically, for the Z' assay, 24 positive control wells (plated at a density of 15,000 cells/well) in 384-well assay plates were challenged with a CFTR-activating mixture of forskolin and IBMX. An equal number of wells were challenged with vehicle alone and containing DMSO (in the absence of activator). Cellular responses in 2 conditions were monitored using a fluorescence plate reader (Hamamatsu FDSS). The mean and standard deviation in 2 conditions were calculated and the mean and standard deviation were calculated using Zhang et al, J Biomol Screen, 4 (2): 67-73(1999) calculates Z'. The Z' value of the resulting stable CFTR expressing cell line was determined to be higher than or equal to 0.82.

Example 14 high throughput screening and identification of CFTR modulators

High-throughput compatible fluorescent membrane potential assays are used to screen for and identify modulators of CFTR. Cells were harvested from the stock plates into antibiotic-free main growth medium 1 day prior to assay and plated into 384-well assay plates with black, clear bottoms. The assay plate was maintained at 37 ℃ in a cell culture incubator at 5% CO₂And the next 19-24 hours. The medium was then removed from the assay plates and added in a loading buffer (137mM NaCl, 5mM KCl, 1.25mM CaCl)₂25mM HEPES, 10mM glucose) and cells were incubated at 37 ℃ for 1 hour. Test compounds were dissolved in dimethyl sulfoxide in assay buffer (137mM sodium gluconate, 5mM potassium gluconate, 1.25mM CaCl)₂25mM HEPES, 10mM glucose) and subsequently loaded into 384-well polypropylene microtiter plates. Cells and compound plates were loaded onto a fluorescent plate reader (Hamamatsu FDSS) and run for 3 minutes to identify test compound activity. The instrument then adds a forskolin solution at a concentration of 300nM-1 μ M to the cells to allow observation of the modulator or blocker activity of the previously added compound. The activity of a compound is determined by measuring the change in fluorescence produced upon addition of the test compound to the cell and/or upon addition of a subsequent agonist.

Example 15 Stable expression for characterization of native CFTR function Using short-circuit Current measurements Cell line of CFTR

The ews laboratory experiment was performed 7-14 days after plating of CFTR expressing cell lines (primary or immortalized epithelial cells, including but not limited to lung and intestine) on culture inserts (Snapwell, corning Life Sciences). Cells on culture inserts were rinsed, fixed on a ewings-type instrument (easysmount Chamber System, PhysiologicInstruments), and treated with a continuously aerated ringer solution (in O) maintained at 37 ℃₂5% CO in₂pH 7.4) bath, the ringer solution comprising 120mM NaCl, 25mM NaHCO₃、3.3mM KH₂PO₄、0.8mM K₂HPO₄、1.2mM CaCl₂、1.2mM MgCl₂And 10mM glucose. The half-cell was connected to a multi-channel potential and current clamp (VCC-MC8, Physiologic Instruments). Use of an electrode [ agar bridged (4% in 1M KCl) -Ag-AgCl]And the insert voltage was clamped to 0 mV. Transepithelial current, voltage and resistance were measured every 10 seconds for the duration of the experiment. The film with a resistance of < 200m omega was discarded.

Example 16 characterization of Stable expression for native CFTR function Using electrophysiological assays Cell line of CFTR

Although both manual and automated electrophysiological assays have been developed and both can be applied to assay such systems, the protocol for the artificial patch clamp experiments is described below.

Cells were seeded at low density and used 2-4 days after plating. Borosilicate glass pipettes were fire polished to obtain tip resistances of 2-4mega Ω. The current is sampled and filtered through a low pass. The extracellular (bathing) solution comprises: 150mM NaCl, 1mM CaCl₂、1mMMgCl₂10mM glucose, 10mM mannitol and 10mM TES, pH 7.4. The pipette solution comprises: 120mM CaCl, 1mM MgCl₂10mM TEA-C1, 0.5mM EGTA, 1mM Mg-ATP and 10mM HEPES (pH 7.3). The membrane conductance was monitored by alternating the membrane potential between-80 mV and-100 mV. By applying a voltage in steps of 20-mV between-100 mV and +100mVThe pulses create a current-voltage relationship.

Example 17 production of Stable cell lines expressing NaV 1.7 heterotrimers Generation of expression constructs

Plasmid expression vectors were generated based on pCMV-script (stratagene) that allow for streamlined cloning and contain various desired components for transcription and translation of genes of interest, including CMV and SV40 eukaryotic promoters; SV40 and HSV-TK polyadenylation sequences; a multiple cloning site; a Kozak sequence and a neomycin/kanamycin resistance cassette (or ampicillin, hygromycin, puromycin, bleomycin resistance cassettes).

Generation of cell lines

Step 1: transfection

293T cells were co-transfected with 3 separate plasmids, one plasmid encoding the human NaV 1.7. alpha. subunit (SEQ ID NO: 19), one plasmid encoding the human NaV 1.7. beta.1 subunit (SEQ ID NO: 20), and one plasmid encoding the human NaV 1.7. beta.2 subunit (SEQ ID NO: 21), using standard techniques. (examples of reagents that can be used to introduce a nucleic acid into a host cell include, but are not limited to, LIPOFECTAMINE^TM、LIPOFECTAMINE^TM 2000、OLIGOFECTAMINE^TM、TFX^TMA reagent,DOTAP/DOPE, metaflecine or FECTURIN^TM。)

Although drug selection is optional for the generation of the cells or cell lines of the invention, we include a drug resistance marker/plasmid. The sequence is under the control of the CMV promoter. Untranslated sequences encoding NaV target sequences for detection by signaling probes are also present along with sequences encoding drug resistance markers. The NaV target sequences utilized were NaV target sequence 1(SEQ ID NO: 22), NaV target sequence 2(SEQ ID NO: 23), and NaV target sequence 3(SEQ ID NO: 24). In this example, the vector containing the NAV 1.7. alpha. subunit gene contains the NaV target sequence 1(SEQ ID NO: 22); the vector comprising the NAV 1.7. beta.1 subunit gene comprises NaV target sequence 2(SEQ ID NO: 23); and the vector comprising the NaV 1.7. beta.2 subunit gene comprises NaV target sequence 3(SEQ ID NO: 24).

Step 2: selecting

The transfected cells were grown in DMEM-FBS medium for 2 days, and then cultured in DMEM-FBS medium containing antibiotics for 10 days. During the period containing the antibiotics, the antibiotics were added to the medium as follows: puromycin (0.1. mu.g/ml), hygromycin (100. mu.g/ml) and bleomycin (200. mu.g/ml).

And step 3: cell passage

After enrichment on antibiotics, cells were passaged 6-18 times in the absence of antibiotics to allow for a time for unstable expression to decline over a selected period of time.

And 4, step 4: contacting the cell with a fluorescent probe

Cells were harvested and transfected with signaling probes (SEQ ID NOS: 25, 26, 27) using standard techniques. (examples of reagents that can be used to introduce a nucleic acid into a host cell include, but are not limited to, LIPOFECTAMINE^TM、LIPOFECTAMINE^TM2000、OLIGOFECTAMINE^TM、TFX^TMA reagent,DOTAP/DOPE, metaflecine or FECTURIN^TM。)

NaV signaling probe 1(SEQ ID NO: 25) binds to NaV target sequence 1(SEQ ID NO: 22);

NaV signaling probe 2(SEQ ID NO: 26) binds to NaV target sequence 2(SEQ ID NO: 23); and NaV signaling probe 3(SEQ ID NO: 27) binds to NaV target sequence 3(SEQ ID NO: 24). Cells were then dissociated and collected for analysis and sorted using a fluorescence activated cell sorter (Beckman Coulter, Miami, FL).

Target sequence detection by a signaling probe

The following target sequences were used for NaV1.7 subunit transgenes.

NaV target sequence 1

5'-GTTCTTAAGGCACAGGAACTGGGAC-3' (SEQ ID NO: 22) (NaV1.7. alpha. subunit)

NaV target sequence 2

5'-GAAGTTAACCCTGTCGTTCTGCGAC-3' (SEQ ID NO: 23) (NaV1.7. beta.1 subunit)

NaV target sequence 3

5'-GTTCTATAGGGTCTGCTTGTCGCTC-3' (SEQ ID NO: 24) (NaV1.7. beta.2 subunit)

Signal conduction probe

Supplied as a 100 μ M stock solution.

NaV signaling probe 1-this probe binds to target sequence 1.

5′-Cy5GCCAGTCCCAGTTCCTGTGCCTTAAGAACCTCGC BHQ3

Quenching-3' (SEQ ID NO: 25)

NaV signaling probe 2-this probe binds to target sequence 2.

5′-Cy5.5CGAGTCGCAGAACGACAGGGTTAACTTCCTCGC BHQ3

Quenching-3' (SEQ ID NO: 26)

NaV signaling probe 3-this probe binds to target sequence 3.

5′-Fam CGAGAGCGACAAGCAGACCCTATAGAACCTCGC BHQ1

Quenching-3' (SEQ ID NO: 27)

The BHQ3 in NaV signaling probes 1 and 2 may be replaced by BHQ2 or gold particles. BHQ1 in NaV signaling probe 3 may be replaced by BHQ2, gold particles, or DABCYL.

Furthermore, those with similar spectral properties to Cy5 were used in specific experimentsDye (BioSearch) similar probes. In some experiments, 5-MedC and 2-aminodA mixmer probes were used instead of DNA probes.

And 5: isolation of Positive cells

Standard analytical methods were used to gate cells that fluoresced above background, and cells that were classified within the defined gate were isolated directly into 96-well plates. Flow cytometry cell sorting operates such that each well holds a single cell. After selection, the cells are expanded in medium lacking the drug.

In cell FAM compared to Cy 5: the following gating levels were used in 0.1-1.0% of live cells:

coincidence gate → unimodal gate → trap door → sorting gate.

Step 6: additional cycles of Steps 1-5 and/or 3-5

Repeating steps 1-5 and/or 3-5 to obtain a larger number of cells. At least 4 independent rounds of steps 1-5 are performed, and for each of these rounds, at least 2 internal rounds of steps 3-5 are performed for each independent round.

And 7: estimation of growth rate of cell population

The plates were transferred to a Microlabstar automated liquid processor (Hamilton Robotics). Cells were incubated for 5-7 days in a 1: 1 mixture of fresh complete growth medium (DMEM/10% FBS) and 2-3 days conditioned growth medium supplemented with 100 units/ml penicillin and 0.1mg/ml streptomycin. The dispersed cells were then trypsinized to minimize clumping and transferred to a new 96-well plate. After clone dispersion, plates were imaged to determine well confluence (Genetix). Each plate is focused for reliable image acquisition across the plate. Not relying on reported confluencies of more than 70%. Confluency measurements were obtained at every 3 days over 9 days (between days 1 and 10 after dispersion) and used to calculate growth rate.

And 8: boxed cell population estimation from growth rate

The cell frames were pooled (grouped independently and plated as contemporaneous groups) according to growth rates between 10-11 days after the dispersion step in step 7. Frames were collected separately and plated on individual 96-well plates for downstream processing; some growth frames produce more than one 96-well plate. Boxes were calculated by considering the spread of growth rate and classifying a high percentage of the total number of cell populations as homogeneous. Depending on the sorting iteration described in step 5, 5-9 growth boxes with 1-4 day separation were used. Thus, each box corresponds to a growth rate or population doubling time difference between 8-14.4 hours, depending on the iteration.

The plates were subjected to standard and fixed conditions (humidified 37 ℃, 50% CO)₂) Next, the cells were incubated in DMEM-10% FBS medium without antibiotics. The plates of cells were separated to generate 4 sets of target plates. These 4 sets of plates included all plates with all growth boxes to ensure that there were 4 replicates of the starting set. Up to 3 target plate sets were submitted for cryopreservation (described in step 10), and the remaining sets were scaled up and further plated repeatedly for passage and for functional assaysAnd (6) testing. Different and independent tissue culture reagents, incubators, personnel and carbon dioxide sources were used for the downstream replica plates. Quality control steps were taken to ensure proper production and quality of all tissue culture reagents: each component added to each bottle of media prepared for use was added by one designated person in one designated fume hood, where only that reagent was present, while the second designated person was supervising to avoid errors. Conditions for liquid handling are set to eliminate cross-contamination between wells. Fresh tips were used for all steps, or a stringent tip washing protocol was used. Liquid handling conditions were set for accurate volume transfer, efficient cell treatment, wash cycles, pipetting speed and positioning, number of pipetting cycles for cell dispersion, and relative position of tip to plate.

Step 10: early passage stock of frozen cell populations

Groups 3 plates were frozen at-70 to-80 ℃. Plates in the set were first allowed to reach 70-80% confluence. The medium was aspirated and 90% FBS and 5% -10% DMSO were added. The plates were sealed with Parafilm, individually surrounded by 1-5cm of foam, and then placed in an 80 ℃ freezer.

The remaining plate sets were maintained as described in step 9. All cells were performed separately using automated liquid processing steps including media removal, cell washing, trypsin addition and incubation, quenching and cell dispersion steps. For some assay plating steps, cells are dissociated with a Cell dissociation buffer (e.g., CDB, Invitrogen or Cell striper, CellGro) instead of trypsin.

The consistency and standardization of cells and culture conditions for all cell populations were controlled. Differences across the plates due to slight differences in growth rates were controlled by regular normalization of the number of cells across the plates per 2-8 passages. Cell populations that are outliers are detected and eliminated.

Step 13: characterization of cell populations

Cells were maintained in cell culture for 3-8 weeks to allow for their evolution in vitro under these conditions. During this time course, size, morphology, fragility, response to trypsin digestion or dissociation, circularity/mean circularity after dissociation, percent viability, tendency towards micro-confluence, or other aspects of cell maintenance such as attachment to the surface of the culture plate were observed.

The cell population is tested using functional criteria. Cells were tested in 96 or 384 well plates at various densities and analyzed for response using a membrane potential assay kit (Molecular Devices/MDS) according to the manufacturer's instructions. Various time points after spreading are used, for example 12 to 48 hours after spreading. Differences in assay response for different coating densities were also tested.

Step 15:

functional responses from experiments performed at low and higher passage numbers were compared to identify cells with the most consistent response over a defined period of 3-9 weeks. Other cellular features that change over time are also noted.

Step 16:

cell populations that meet functional and other criteria are further evaluated to determine those that are most amenable to the generation of viable, stable and functional cell lines. The selected cell population is expanded in a larger tissue culture vessel and the characterization steps described above are continued or repeated under these conditions. In this regard, additional normalization steps are introduced, such as different plated cell densities; passage time; petri dish size/form and coating); optimizing the fluidics; cell dissociation optimization (e.g., type; volume and length of time used); and a washing step for consistent and reliable passage. The temperature difference is also used for normalization (e.g. 30 ℃ compared to 37 ℃).

And step 17: establishment of cell banks

The low passage freezing plates described above corresponding to the final and backup cell lines were thawed at 37 ℃, washed 2 times with DMEM-10% FBS, and humidified at 37 ℃/5% CO₂Incubation under conditions. The cells were subsequently expanded for a period of 2-3 weeks. A cell bank was established for each final and backup cell line, consisting of 15-20 vials.

Step 18:

the following steps may also be performed to confirm that the cell line is viable, stable and functional. At least one vial from the cell bank is thawed and expanded in culture. The resulting cells are tested to determine whether they meet the same characteristics according to which they were originally selected.

Example 18 characterization of heterologous NaV1.7 Subsomonas in Stable NaV1.7 expressing cell lines Relative expression of bits

Quantitative RT-PCR (qRT-PCR) was used to determine the relative expression of heterologous human NaV1.7 α, β 1 and β 2 subunits in the resulting stable NaV1.7 expressing cell lines. Purification from 1-3X 10 Using RNA extraction kit (RNeasy MiniKit, Qiagen) ⁶Total RNA of individual mammalian cells. The DNase treatment was done according to a strict DNase treatment protocol (TURBO DNA-freeKit, Ambion). Using the reverse transcription kit (SuperScript III, Invitrogen), the DNA-free total RNA was added at a concentration of 1. mu.gFirst strand cDNA synthesis was performed in a 20 μ L reaction volume of 250ng random primer (Invitrogen). Samples without reverse transcriptase and samples without RNA were used as negative controls for this reaction. The synthesis was carried out in a thermocycler (Mastercycler, Eppendorf) under the following conditions: 5 minutes at 25 ℃ and 60 minutes at 50 ℃; the reaction was terminated at 70 ℃ for 15 minutes.

For the analysis of gene expression, primers and probes for qRT-PCR (MGBTaqMan probe, Applied Biosystems) were designed to anneal specifically to the target sequences (SEQ ID NOS: 22, 23, 24). For sample normalization, a control (glyceraldehyde 3-phosphate dehydrogenase (GAPDH)) Pre-Developed Assay reagent (TaqMaN, Applied Biosystems) was used. Reactions including negative and positive controls (plasmid DNA) were set up in triplicate using 40ng of cDNA in a 50 μ L reaction volume. The relative amounts of each of the 3 NaV 1.7 subunits expressed were determined. All 3 subunits were successfully expressed in the generated stable NaV 1.7 expressing cell line.

Example 19 characterization of stable NaV 1.7 expressing cell lines for native NaV function Using electrophysiological assays

An automated patch clamp system was used to record sodium currents from the generated stable HEK293T cell line expressing NaV 1.7 α, β 1 and β 2 subunits. The following exemplary scheme may also be used for QPatch, Sophion or Patchliner, Nanion systems. At room temperature, the extracellular ringer solution contained 140mM NaCl, 4.7mM KCl, 2.6mM MgCl₂11mM glucose and 5mM HEPES, pH 7.4. The intracellular ringer solution comprises 120mM CsF, 20mM Cs-EGTA, 1mM CaCl₂，1mM MgCl₂And 10mM HEPES, pH 7.2. The experiments were performed at room temperature.

Cells stably expressing NaV 1.7 α, β 1 and β 2 subunits were grown under standard culture protocols as described in example 17. Cells were harvested and kept suspended with continuous stirring for up to 4 hours with no significant change in the quality or capacity of the membrane. Electrophysiological experiments (whole cells) were performed using standard patch plates. The patch clamp holes (microetched in the chip) are about 1 μ M in diameter and have a resistance of 2M Ω. The membrane potential was clamped to a holding potential of-100 mV.

Characterization of the current-voltage relationship and inactivation characteristics of the voltage-gated human NaV 1.7 sodium channel stably expressed in HEK293T cells. The sodium current was measured in response to a 20ms depolarization pulse from-80 mV to +50mV with a holding potential of-100 mV. The resulting current-voltage (I-V) relationship for the peak sodium channel current was characterized. The activation threshold was-35 mV (midpoint of activation, Va ═ 24.9mV +/-3.7mV)), and the maximum current amplitude was obtained at-10 mV. Inactivation patterns for sodium channels were plotted. The membrane potential was held at a holding potential of-100 mV, followed by a transition to a conditioning potential of +10mV from-110 mV for 1000ms, and finally the current was measured after a step to 0 mV. The resulting current amplitude is indicative of the portion of the sodium channel that is in the inactive state. At potentials more negative than-85 mV, the channels are predominantly in the closed state, whereas at potentials above-50 mV, they are predominantly in the deactivated state. The curves represent Boltzmann fits according to which V is related to steady state inactivation _1/2Estimated to be-74 mV. Current-voltage spectra for the resulting stable NaV 1.7 expressing cell lines and the previously reported current-voltage spectra (Va ═ 28.0 mV. + -. 1.1 mV; V)_1/2-71.3mV ± 0.8mV) (Sheets et al, J physiol.581(Pt 3): 1019 and 1031. (2007)).

Example 20 characterization of Stable expression of NaV 1.7 for native NaV function Using Membrane potential assay Cell line of (2)

The resulting stable NaV 1.7 α, β 1 and β 2 subunit-expressing cells were maintained under standard cell culture conditions in Darbeke's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, glutamine and HEPES. Cells were harvested from the seed plates using cell dissociation buffer such as cdb (gibco) or cell stripper (Mediatech) 1 day prior to assay and plated at 10,000-25,000 cells/well into growth medium in 384-well assay plates. The assay plate was maintained at 37 ℃ in a cell culture incubator at 5% CO₂And the next 22-24 hours. Followed by measuring the levelThe plate medium was removed and buffer loaded (137mM NaCl, 5mM KCl, 1.25mM CaCl)₂25 mhepes, 10mM glucose) was added to the sample. Cells were incubated with blue membrane potential dye for 1 hour at 37 ℃. The assay plates were then loaded onto a high throughput fluorescence plate reader (hamastu FDSS). The fluorescence plate reader measures the cell fluorescence in the image of the cell plate obtained once per second and displays the data as relative fluorescence units.

The assay response of stable NaV 1.7 expressing cells and control cells (i.e., HEK293T parental cells) to the addition of buffers and channel activators (i.e., veratridine and Scorpion Venom (SV)) was measured. In the first addition step (i.e., addition 1), only buffer was added, and no test compound was added. If desired, the test compound may be added during this step. In the second addition step, veratridine and scorpion venom, which are sodium channel activators, are diluted to the desired concentration in assay buffer (i.e., 25 μ M veratridine and 5-25 μ g/ml scorpion venom) and added to 384-well polypropylene microtiter plates. Upon binding, veratridine and scorpion venom proteins modulate the activity of voltage-gated sodium channels by a combination of mechanisms, including changes in activation and inactivation kinetics. The resulting activation of sodium channels in stably NaV 1.7 expressing cells alters the cell membrane potential and the fluorescence signal increases. The above functional assays can also be used to characterize the relative potency of test compounds for NaV 1.7 ion channels.

Example 21 characterization of NaV 1.7a subunit Regulation by the B subunit

Modulation of alpha subunit gene expression by beta subunit

Pools of HEK293T cells were engineered to express a variety of ratios of alpha and beta subunits by treating individual plasmid DNA or the molar ratio of alpha to control plasmid (e.g. alpha: beta 1: beta 2 ═ 1: 1). After drug selection, subunit expression in 6 different cell depots was assessed using qRT-PCR as described in example 18. Comparative qRT-PCR indicated that when all 3 human NaV 1.7 subunits (i.e., α, β 1 and β 2) were co-transfected, the expression of the α subunit increased in drug-selected cell assays compared to the α subunit alone and the control plasmid. The presence of the beta subunit transcript affects alpha subunit gene expression, confirming the importance of co-expressing all 3 NaV 1.7 subunits for physiologically relevant functional assays.

Modulation of pharmacological properties by beta subunit

Cell-based membrane potential assays were used to measure the response to test compounds of cells stably co-expressing all 3 NaV1.7 subunits (i.e., alpha, beta 1, and beta 2), and control cells stably expressing only NaV1.7 alpha subunits. 2 compounds (i.e., C18 and K21) were tested in membrane potential assays performed simultaneously according to the protocol in example 20. In particular, for this example, the test compound was added in the first addition step.

C18 and K21 enhanced the response of clone C44 (expressing NaV1.7 α, β 1 and β 2 subunits) and blocked the response of clone C60 (expressing NaV1.7 α subunit only). For each of the 2 clones, the assay responses of the 2 test compounds were normalized against the response of the individual buffers.

Example 22 characterization of different subunit combinations of NaV1.7

Membrane potential cell-based assays were used to measure the response to test compounds to different cell lines stably co-expressing all 3 nav1.7 subunits (i.e., α, β 1 and β 2). Dose-response analysis (DRC) of a panel of compounds tested on 4 cell lines generated from cells positive for expression of NaV1.7 α, β 1 and β 2 subunits resulted in different functional profiles (fig. 4). It was found that a plurality of cell lines contained a signature profile similar to each of the 4 signature profiles shown in fig. 4.

Based on their functional pharmacological properties in a cell-based assay, approximately 90 cell lines co-expressing all 3 NaV 1.7 subunits (i.e., α, β 1 and β 2) were classified, with each clonal cell line tested using the same compound (fig. 5). Each cluster in FIG. 5 (boxes 1-5) represents a subset of clones of a particular similar activity. Activity was calculated as the percentage of maximal signal inhibition as shown in figure 5 for each clonal cell line. The negative numbers in fig. 5 indicate the percentage of enhancement.

TABLE 7 mammalian G proteins, their families and descriptions

Note that: the cells may express various combinations of any of such proteins.

TABLE 8 human orphan GPCRs, including their Gene symbol and NCBI Gene ID numbering

TABLE 9 List of human opioid receptors

TABLE 10 list of human olfactory receptors

TABLE 11 List of canine olfactory receptors (their Gene names)

TABLE 12 Anopheles gambiae olfactory receptors

Note that: GPRor7/IOR55 can be co-expressed with each of the other ORs.

TABLE 13 receptor List

Note that: cells can also be prepared using corresponding sequences from other species; this is an exemplary list of classes of receptors, not all families or family members are listed, and other receptors can also be expressed in cells using our method.

TABLE 14 GABA subunits from different species

TABLE 15 list of human bitter taste receptors

Preferred G proteins for use in preparing bitter taste receptor cell lines include, but are not limited to, mouse G α 15 and human GNA 15.

TABLE 16 sweet and umami taste receptors

TABLE 17 cystic fibrosis transmembrane conductance regulator

TABLE 18 guanylate cyclase

TABLE 19 mouse olfactory receptor List

TABLE 20 polymorphisms with the human bitter taste receptor gene

(Table 20 contains the sequence number of each unique Single Nucleotide Polymorphism (SNP) of the human TAS2R gene, the position of the SNP in the reference sequence, and a description of the SNP.)

TABLE 21 allelic variation in the coding sequence of human bitter taste receptors

Note that: all positional information in table 21 refers to the canonical sequence of human TAS2R protein.

TABLE 22 insect Gene List

Human odorant receptor homologs of the receptors listed in table 22 may also be used with the methods and compositions of the present invention.

Example 23 Generation of Stable cell lines expressing the umami receptor

Transfection

HEK293T (ATCC CRL-11268) was co-transfected with 3 separate plasmids, one encoding T1R1(SEQ ID NO: 41), one encoding T1R3(SEQ ID NO: 32) and the other encoding a signaling molecule (mouse G.alpha.15, SEQ ID NO: 33). Although drug selection is optional in the methods of the invention, we include a drug resistance marker/plasmid. The sequence is under the control of the CMV promoter. The untranslated sequence encoding the tag for detection by the signaling probe is also present along with the sequence encoding the drug resistance marker. The target sequences utilized were target sequence 1(SEQ ID NO: 28), target sequence 2(SEQ ID NO: 29) and target sequence 3(SEQ ID NO: 30). In these examples, the T1R1 gene vector comprises target sequence 3, the T1R3 gene vector comprises target sequence 1, and the ga 15 gene vector comprises target sequence 2. Cells are routinely selected in media containing drugs for 10-14 days.

Contacting the cell with a fluorescent probe

Selected cells were harvested and transfected with the signaling probe (SEQ ID NOS: 38-40). Signaling probe 1 binds to tag sequence 1(SEQ ID NO: 126), signaling probe 2 binds to tag sequence 2(SEQ ID NO: 127) and signaling probe 3 binds to tag sequence 3(SEQ ID NO: 128). Cells were then dissociated and collected for analysis and sorted using a fluorescence activated cell sorter (Beckman Coulter, Miami, FL). As will be appreciated by one of ordinary skill in the art, any agent suitable for use with the selected host cell can be used to introduce nucleic acids (e.g., plasmids, oligonucleotides, labeled oligonucleotides) into the host cell with appropriate optimization. Examples of reagents that can be used to introduce nucleic acids into a host cell include, but are not limited to, Lipofectamine 2000, Oligofectamine, TFX reagens, Fugene 6, DOTAP/DOPE, Metafectine, or Fecturin.

Signal transduction probe 1

5 '-Cy 5 GCCAGTCCCAGTTTCCTGTGTGCCTTAAGAACCTCGC BHQ3 quenching-3'

(SEQ ID NO：38)

Signal transduction probe 2

5 '-Cy5.5GCGAGTCGCAGAACGACAGGGTTAACTTCCTCGCBHQ 3 quenching-3'

(SEQ ID NO：39)

Signal transduction probe 3

5 '-Fam GCGAGAGCGACAAGCAGACCCTATAGAAACCTCGCCBHQ 1 quenched-3'

(SEQ ID NO：40)

Target sequence 1, target sequence in bold (umami)

AGGGCGAATTCCAGCACACTGGCGGCCGTTACTAGTGGATCCGAGCTCGGAGGCAGGTGGACAGGAAGGTTCTAATCTGGGCCCGGAAAGCCTTTTTCTCTGTGATCCGGTACAGTCCTTCTGC (SEQ ID NO：126)

Target sequence 2, target sequence in bold (umami)

AGGGCGAATTCCAGCACACTGGCGGCCGTTACTAGTGGATCCGAGCTCGGTACCAAGCTTCGAGGCAGGTGGACAGCTTGGTTCTAATATCTGGGCCCGGAAAGCGTTTAACTGATGGATGGAACAGTCCTTCT GC(SEQ ID NO：127)

Target sequence 3, target sequence in bold (umami)

AAGGGCGAATTCGGATCCGCGGCCGCCTTAAGCTCGAGGCAGGTGGACAGGAAGGTTCTAATATCTGGGCCCGGAGATG(SEQ ID NO：128)

Other target sequences and signaling probes can be used (see, e.g., International patent application publication No. WO2005/079462 (application No. PCT/US 05/005080)) as published on 9/1/2005. For example, BHQ3 may be replaced by BHQ1 or gold particles in probe 1 or probe 2. It should be noted that BHQ1 can be replaced by BHQ2 or Dabcyl in Probe 3. Similar probes using Quasar Dye (BioSearch) with similar spectral properties as Cy5 can be used in certain experiments. It should also be noted that in some cases 5-MedC and 2-aminodA mixmer probes are used instead of DNA probes.

Isolation of Positive cells

Standard assay methods are used to gate cells that fluoresce above background and isolate individual cells that fall within the gate into a barcoded 96-well plate. Cell sorting was operated so that a single cell was placed per well. After selection, the cells are expanded in medium in the absence of the drug.

Functional transformation

We maintained the umami receptor cells (selected and expanded as above) in various growth conditions including, for example, low glucose DMEM medium or in glucose-free Leibovitz L-15 medium with 10% serum or serum-free using two aliquots of the same cells and different cells. Some cells were maintained in media containing different concentrations of other sugars, such as galactose. We then characterized cells of the umami receptor cell line maintained under a variety of conditions for their ability to respond appropriately to umami ligands and not to respond to other stimuli (i.e., sugars).

Without wishing to be bound by theory, growth in different media conditions may functionally transform cells, for example, due to changes in gene expression levels, genomic tissue, and functional expression of receptors on the cell surface. Parameters in different media evaluated and shown to affect the functional assay response of cells include serum concentration (i.e., low serum concentration and serum deprivation), sugar deprivation, and assay plate coating (e.g., poly-D-lysine and laminin). These results show that the characteristics of cells of the umami receptor cell line can be different when the cells are maintained in different media conditions. They also show that different cells selected by fluorescent probes may have different characteristics. An example of our characterization of cells selected by fluorescent probes is shown in figure 6. As shown in figure 6, aliquots of the same cells cultured in different conditions (1, 2 and final) responded significantly differently to umami (MSG) and sweet (fructose) ligands. The cells cultured in condition 1 were plated in a low glucose medium deprived of serum. The cells cultured in condition 2 were plated overnight in low glucose medium containing 10% serum and then switched to serum deprived medium before measurement. Cells cultured in condition 1 or 2 were plated on a coated plate (Corning # 3665). "Final" conditions included high density cultures on coated plates (Corning #3300) and low glucose media with serum deprivation. The cells cultured in condition 1 respond to the sweet taste agonist more than to the umami taste agonist. Cells cultured in condition 2 responded equally to both ligands. In contrast, cells cultured in "final" culture conditions responded strongly to MSG but not fructose. Thus, these cells produce physiologically and pharmacologically relevant umami receptors.

Example 24 characterization of cell lines for Natural umami receptor function

1. Verification and quantification of gene expression

By using qRT-PCR, we determined the relative amount (RNA) of each umami receptor subunit expressed in the above cells ("final"). By using qRT-PCR, we determined the relative amount of each umami receptor subunit expressed. From 1-3X 10 using a commercially available RNA purification Kit (RNeasy Mini Kit, Qiagen)⁶Total RNA was purified from individual mammalian cells. The RNA extract was then treated using a stringent DNase treatment protocol (TURBO DNA-free Kit; Ambion). First strand cDNA synthesis was performed in 20. mu.L using the Reverse transcription kit (Superscript III, Invitrogen) using 1. mu.g of total RNA without DNA and 250nG random primers (Invitrogen). Negative controls for this reaction include samples in which reverse transcriptase or RNA is not added during the cDNA synthesis step. cDNA and PCR product synthesis was performed in a thermal cycler (Mastercycler Eppendorf) under the following conditions: at 25 ℃ for 5 minutes and at 50 ℃ for 60 minutes; the reaction was terminated at 70 ℃ for 15 minutes.

To analyze gene expression (RNA), probes for T1R1, T1R3, and mouse G.alpha.15 cDNA (MGB TaqMan probes, Applied Biosystems) were used. For the sample normalization control, GAPDH, Pre-Developed TaqMaN Assay Reagents, Applied Biosystems were used. Reactions were set up in triplicate in a 50 μ L reaction volume using 40ng of cDNA, including negative and positive controls (plasmid DNA). The relative amount of each umami receptor subunit expressed (RNA) is determined. Figure 7 graphically depicts the results and shows that all 3 nucleic acids are expressed in the umami receptor cell line (RNA). Expression levels of T1R1, T1R3, and ga 15 were approximately 10,000, 100, and 100,000 times the levels observed in control cells, respectively.

Standard single-endpoint RT-PCR methods were used to estimate gene expression (RNA) for T1R1, T1R3, and ga 15 in 1 and 9 month cultures producing umami receptor cell lines according to the protocol described above ("final"). Cells were cultured in a 24-well plate format to 80% confluence, harvested and RNA isolated using a commercially available RNA preparation kit (RNAqueous kit, Ambion). Purified total RNA in the range of 5pg to 5. mu.g was used for reverse transcription using oligo (dT)12, 18 primers according to the protocol of a commercial first strand cDNA synthesis kit (Superscript III kit, Invitrogen). Following first strand synthesis, nucleic acids specific for subunits of umami receptor (T1R1, T1R3) and the nucleic acid group of mouse G α 15 were independently assembled in a PCR reaction mixture (HotStart Taq). After 45 cycles of PCR, the amplicon samples were further analyzed using agarose gel electrophoresis.

The results from these single-endpoint RT-PCR experiments are illustrated in fig. 8. FIG. 8 depicts representative photographs of agarose gels used for RT-PCR experiments. Strong expression (RNA) of all nucleic acids encoding umami receptors was detected in cultures of 1 month and longer, 9 months, demonstrating an abnormal level of stability of the cell lines of the invention cultured under "final" conditions.

2. Cell-based assays for modulators

The cells of the invention were cultured at (75-125K)/well 24 hours prior to the assayMedia (low glucose DMEM or L15 media supplemented with serum and standard growth additives) were seeded in 96-well plates. After incubation, the growth medium was removed and the cells were placed in serum-free growth medium. Cells were incubated for 2-3 hours. The medium was then removed, and the cells were then loaded with calcium sensitive fluorescent dye (calcium-3, Molecular Devices Corp.) diluted in umami taste assay buffer (130mM NaCl, 1.1mM KH)₂PO₄1.3mM CaCl, 20mM HEPES and 3mM NaHPO₄*7H₂O). Cells were incubated in this medium for 1 hour. Plates were loaded onto a high-throughput fluorescence plate reader (Hamamatsu FDSS). Test compounds were diluted to the desired concentration in umami assay buffer and added to each well. Calcium flux was measured for 90 seconds. Activator (i.e., MSG) diluted in the above buffer was added to each well at a final concentration ranging between 10. mu.M to 100mM, and the change in relative fluorescence was recorded for an additional 90 seconds.

3. Determination of Z' and EC according to Umami taste cell-based assay ₅₀ Value of

To test the efficacy of the umami receptor response in such umami receptor expressing cell lines, the established umami receptor activator monosodium glutamate (MSG) was used as a test compound in the above assay. MSG (Sigma, G5889) was added to the test wells at a concentration of 33mM and the control wells received buffer only. In the final assay conditions, as reported by calcium flux measurements measured by a fluorescent plate reader. (FLIPR3 operating System, Molecular Devices). In one assay repeated several times, the cell line had a Z' value of 0.8. See fig. 9 (an exemplary assay). The Z' value indicates that the resulting cells expressing the umami receptor recognize MSG in cell-based assays, and that such assays can be reliably and stably performed using such cells.

To test the sensitivity of the umami receptor response in the umami receptor expressing cell lines of the invention, the cells were dosed with increasing amounts of MSG, followed by the same procedure as described aboveThe responses were measured to perform a dose response experiment. In one assay (FIG. 10), the EC of MSG in this cell line was found₅₀The value was 22 mM. These results indicate that the umami receptor produced in the cell line of the invention exhibits strong sensitivity to known umami receptor ligands in the cell line of the invention expressing umami receptor.

EXAMPLE 25 known enhancers IMP and sodium Cyclohexylsulfamate enhance the umami taste cell line Reaction to MSG

As mentioned above, MSG is a known umami receptor ligand. The nucleotide inosine monophosphate (IMP, Sigma) has also been shown to be useful as an enhancer of umami receptor signaling when presented together with MSG. By using the above assay methods, matrix arrays of increasing MSG and increasing IMP concentrations can be applied to cell lines and responses generated. Figure 11 shows that for one such assay, as IMP concentration increases, a stronger response is detected at each concentration of MSG tested.

Similar to MSG, sodium cyclamate (artificial sweetener) can be used as an activator of the umami receptor when it interacts with a subunit common to both sweet and umami receptors. By using the above assay protocol, substrates of increasing cyclohexylsulfamate (Sigma) concentration were utilized and reactions were generated. Figure 12 shows that for one assay, more significant reactions were detected as the cyclohexylsulfamate concentration increased. This is in contrast to several other cell lines described in the prior art, which did not detect cyclohexylsulfamate in the absence of MSG.

Example 26 Generation of Stable bitter taste receptor expressing cell lines

Step 1-transformation

293T cells were co-transfected with 2 separate plasmids, one encoding the TAS2R bitter taste receptor (one of SEQ ID NO: 77-101) and the other encoding the mouse G.alpha.15 signaling protein (SEQ ID NO: 102). As will be appreciated by one of ordinary skill in the art, any agent suitable for use with the selected host cell can be used to introduce nucleic acids (e.g., plasmids, oligonucleotides, labeled oligonucleotides) into the host cell with appropriate optimization. Examples of agents that can be used to introduce nucleic acids into a host cell include, but are not limited to, Lipofectamine 2000, Oligofectamine, TFX agents, Fugene 6, DOTAP/DOPE, Metafectine, or F ecturin. Although drug selection is optional in the methods of the invention, we include a mammalian drug-resistance marker/plasmid. The plasmid uses the CMV promoter for expression of the bitter taste receptor gene or the G α 15 gene. The untranslated sequence encoding the tag for detection by the signaling probe is also present in each vector along with the sequence encoding the drug resistance marker, such that the tag is transcribed along with the protein expressed by the vector, i.e., the bitter taste receptor or G α 15. The target sequences utilized were target sequence 1(SEQ ID NO: 46), target sequence 2(SEQ ID NO: 47). In these examples, the vector comprising the TAS2R gene comprises target sequence 1 and the vector comprising the ga 15 gene comprises target sequence 2.

Step 2: selection step

The transfected cells were cultured in Darbevaceae Modified Eagle Medium (DMEM) containing Fetal Bovine Serum (FBS) for 2 days, followed by DMEM-FBS containing antibiotics for 14 days. The antibiotic-containing period had the following antibiotics added to the medium: puromycin (0.15. mu.g/ml) and hygromycin (100. mu.g/ml).

And step 3: cell passage

After enrichment on antibiotics, and prior to introduction of fluorescent signaling probes, cells were passaged 8(p5-p13) more times in the absence of antibiotics to allow time for unstable expression to decline over a selected period of time.

And 4, step 4: contacting the cell with a fluorescent probe

Cells were harvested and transfected with signaling probes (SEQ ID NOS: 48 and 49). As will be appreciated by one of ordinary skill in the art, any agent suitable for use with the selected host cell can be used to introduce nucleic acids (e.g., plasmids, oligonucleotides, labeled oligonucleotides) into the host cell with appropriate optimization. Examples of reagents that can be used to introduce nucleic acids into a host cell include, but are not limited to, Lipofectamine 2000, Oligofectamine, TFX reagens, Fugene 6, DOTAP/DOPE, Metafectine, or F ecturin.

Target sequence detection by a signaling probe

Target 1

5′-GTTCTTAAGGCACAGGAACTGGGAC-3′(SEQ ID NO：46)

Target 2

5′-GAAGTTAACCCTGTCGTTCTGCGAC-3′(SEQ ID NO：47)

Similar probes of quasardye (biosearch) with similar spectral properties as Cy5 were used in specific experiments. It should also be noted that in some cases 5-MedC and 2-aminodAmixmer probes are used instead of DNA probes.

Random non-targeting fam probes were used as delivery controls (not shown).

Signal conduction probe

The signaling probe was supplied as a 100. mu.M stock solution.

Signaling probe 1-binding target 1

5 '-Cy 5 GCCAGTCCCAGTTTCCTGTGTGCCTTAAGACCTCGCBHQ 3 quenching-3' (SEQ ID NO: 48)

Signaling probe 2-binding target 2

5 '-Cy5.5GCGAGTCGCAGAACGACAGGGTTAACTTCCTCGCBHQ3 quenching-3' (SEQ ID NO: 49)

It should be noted that BHQ3 can be replaced by BHQ1 or gold particles in Probe 1 or Probe 2.

BHQ3 can be replaced by BHQ2 or gold particles in probe 1 or probe 2.

And 5: isolation of Positive cells

Cells were dissociated and collected for analysis and sorted using a fluorescence activated cell sorter (Beckman Coulter, Miami, FL). Standard assay methods are used to gate cells that fluoresce above background and isolate individual cells that fall within the gate into a barcoded 96-well plate. The gating levels are as follows: coincidence gate > unimodal gate > valve > sorting gate. With this gating strategy, the top 0.1-1.3% of double positive cells were marked for sorting into barcoded 96-well plates.

Step 6: additional cycles of Steps 1-5 and/or 3-5

The experiment was staged with very compact timing logic. As part of this movement, there are two full cycles of steps 3-5 that are performed to complete the redundant sorting and to obtain additional clones.

And 7: estimation of growth rate of cell population

The plates were transferred to a Hamilton Microlabstar automated liquid handler. Cells were grown on 2-3 day conditions in medium: incubation in a 1: 1 mixture of fresh growth medium (DMEM/10% FBS) supplemented with 100 units penicillin/ml plus 0.1mg/ml streptomycin for up to 20 days. Plates were imaged 7 to 20 days after sorting to determine well confluence (Genetix). Each plate is focused for reliable image acquisition across the plate. Not relying on reported confluencies of more than 70%. Confluent measurements were obtained 3 times over the time specified above and used to calculate growth rate.

And 8: boxed cell population estimation from growth rate

The cells were framed (grouped independently and plated as contemporaneous clusters) according to a growth rate of about 20 days after sorting. Frames are collected independently and plated on individual 96-well plates for downstream processing, and there may be more than one target plate/specific frame. The boxes were calculated by considering the spread in growth rate and classifying ranges covering a high percentage (at least about 90% (as an estimate)) of the total number of cell populations as homogeneous. 5 growth boxes with an average separation of 1.2-3.5 days were used in boxes spanning different bitter receptor cell lines. Thus, each box corresponds to a growth rate or population doubling time difference at about 11 hours.

Cells may have doubling times of less than 1 day to over 2 weeks-in order to process the most diverse clones that can be simultaneously binned reasonably according to growth rate, we generally prefer to generate 3-9 bins at 0.25-0.7 day doubling times per bin. One of ordinary skill in the art would understand how to adjust the tightness of the boxes and the number of boxes on a case-by-case basis, and if the cells are synchronized according to their cell cycle, the tightness and number of boxes may be further adjusted.

Step 9-repeat coating to speed parallel processing and provide tight quality control

The plates were subjected to standard and fixed conditions (humidified 37 ℃, 5% CO)₂) Incubation in DMEM medium/10% FBS without antibiotics follows. The plates of cells were separated to generate 4 sets (a set consisting of all plates with all growth frames, these steps ensuring that there were 4 replicates of the starting set) of target plates. Up to 3 target plate groups were submitted for cryopreservation (see below), and the remaining groups were scaled up and further replated for passage and for functional assays experiments. Different and independent tissue culture reagents, incubators, personnel and carbon dioxide sources were used for each set of plates. Quality control steps were taken to ensure proper production and quality of all tissue culture reagents: each component added to each bottle of media prepared for use was added by one designated person in one designated fume hood, where only that reagent was present, while the second designated person was supervising to avoid errors. Conditions for liquid handling are set to eliminate cross-contamination between wells. Fresh tips were used for all steps, or a stringent tip washing protocol was used. Setting liquid treatment Conditions are used for accurate volume transfer, efficient cell treatment, wash cycles, pipetting speed and positioning, number of pipetting cycles for cell dispersion, and relative position of tip to plate.

Step 10: early passage stock of frozen cell populations

Groups 3 plates were frozen at-70 to-80 ℃. Plates in each group were first allowed to reach 70 to 100% confluence. The medium was aspirated and 90% FBS and 10% DMSO were added. The plates were sealed using Parafilm and then surrounded with 1 to 5cm of foam, respectively, and placed into a-80 ℃ freezer.

Step 11: methods and conditions for an initial transformation step to generate stable cell lines

Step 12: testing of functionality of cells in a rearranged plate

Due to the size of the recipient cell object, the functionality of the growth rearranged plates was not used as a first priority (between 3-5 passages after rearrangement) but rather was quickly determined and a subset population of responders for each of the 25 bitter taste receptors was identified.

Step 13: characterization of cell populations

Clones were screened between 3.5 to 6 weeks after sorting and the apical clones were retested functionally and identified between 5 to 6 weeks after sorting. Cells were maintained for up to 6 weeks in cell culture to allow for their evolution in vitro under these conditions. During this time course, we observed size, morphology, fragility, response to trypsin digestion or dissociation, circularity/mean circularity after dissociation, percent viability, tendency towards micro-confluence, or other aspects of cell maintenance such as attachment to the culture plate surface.

Step 14: assessment of potential functionality of cell populations

The cell population is tested using functional criteria. The calcium mobilization dye kit (calcium 3, Molecular Devices/MDS) was used according to the manufacturer's instructions. Cells were tested in 96 or 384 well plates at various densities and analyzed for response. Various time points after spreading are used, for example 12 to 48 hours after spreading. Differences in assay response for different coating densities were also tested.

Functional responses from experiments performed at low or higher passage numbers were compared to identify cells with the most consistent response over a defined period of 6-11 weeks after sorting. It is also noted that other cellular features that change over time, such as the time for cell detachment followed by reattachment, are noted.

Step 16-further evaluation of cells

Cell populations that meet functional and other criteria are further evaluated to determine those most amenable to the production of viable, stable, and functional cell lines. The selected cell population is expanded in larger tissue culture dishes and the characterization steps described above are continued or repeated under these conditions. In this regard, additional standardization steps were introduced for consistent and reliable passaging. These include different plated cell densities, plate coating, passage times, dish size/form and coating, fluidics optimization, cell dissociation optimization (e.g., dissociation in the presence of cell dissociation buffer (Invitrogen) for trypsin), volume and length of time of dissociation reagents used, and washing steps. Glutamine concentration is the dosage range for the medium. In addition, cell viability at each passage was determined. Increasing manual intervention and more closely observing and monitoring the cells. This information is used to help identify and select the final cell line that retains the desired properties. Final and backup cell lines showing consistent growth, consistent (i.e., no morphological changes) proper attachment, and functional response were selected.

And step 17: establishment of cell banks

Low passage freezing plates (see above) corresponding to the final and backup cell lines were thawed at 37 ℃, washed 2 times with DMEM/10% FBS, and humidified at 37 ℃/5% CO₂Incubation under conditions. The cells were subsequently expanded for a period of 2-3 weeks. Cell banks were established for each final and backup cell line, each cell line bank consisting of 25 vials. Cells were cryopreserved in 50% DMEM/10% FBS, 40% FBS and 10% DMSO for further optimization experiments.

Step 18: testing of cell banks

At least one vial from a cell bank (including frozen stock solution of thawed cell lines that have been expanded and refrozen) is thawed and expanded in culture. The resulting cells were tested to confirm that they met the same characteristics according to which they were originally selected.

Example 27 characterization of cell lines for Natural bitter taste receptor function

To identify and measure ligand-induced responses of HEK293 cells expressing bitter taste receptors, receptor activation was monitored by measuring changes in intracellular calcium levels triggered by the receptors. Cells were cultured overnight in standard growth media in black clear bottom plates or as assay buffer (10mM HEPES, 130mM NaCl, 2mM CaCl) ₂，5mM KCl，1.2mMMgCl₂10mM glucose, pH 7.4) was added to a black transparent bottom plate. Cells were incubated with the wash-free calcium sensitive fluorescent dye calcium-3 (molecular devices Corp.) in buffer for 1 hour at room temperature. The cell plates and test compounds (0.01. mu.M-100 mM) were placed in a high throughput fluorescence plate reader (Hamamatsu FDSS) that collected images of plate fluorescence before, during, and after the instrument added the test compounds to the cells. Image software (Hamamatsu FDSS) analysis reported changes in relative fluorescence for each well in the cell plate.

A variety of compounds and extracts (many of which were reported to be bitter) were assayed across 25 bitter receptor cell lines as well as control cells to determine activity and arc-off.

Example 28 functional differences between transiently transfected native and labeled receptors

Transient transfection of native and labeled receptors prior to performing the assay allows analysis of functional differences between native and labeled receptors. As an example, functional assays were performed in Human Embryonic Kidney (HEK)293T cells 48 hours after transient transfection of the mouse ga 15 protein and either the native T2R16 bitter taste receptor or the same receptor with an N-terminal tag from the mouse rhodopsin gene. Cells were incubated with buffer (10mM HEPES, 130mM NaCl, 2mM CaCl) at room temperature ₂、5mM KCl、1.2mM MgCl₂10mM glucose, pH 7.4) for 1 hour. Assay reactions for bitter taste receptor activity were measured in a Hamamatsu FDSS fluorescence plate reader, which collected images of plate fluorescence before, during, and after the instrument added a range of concentrations (0.01 μ M to 100 μ M) of bitter extracts. The FDSS software analyzes the images and reports changes in relative fluorescence for each well in the cell plate.

The results are plotted in fig. 13. As shown in fig. 13, bitter extracts selectively activated natural bitter receptors to a greater extent than the rhodopsin tag-containing receptors. This result suggests that the native human bitter taste receptor and the tagged human bitter taste receptor exhibit different functional activities, and underscores the importance of assays based on cells expressing the native protein and being physiologically relevant for proper receptor assignment and deselection.

Example 29 high success rate of Generation of functional clones of bitter taste receptors

The success rate (number of clones expressing functionally active receptors in all isolated clones) is a crucial parameter for determining the efficacy of generating stable cell lines. We performed a functional assay of the clones we isolated using the method described in example 26 and found that more than 80% of the isolated clones had functional activity.

As an example, individual clonal cell lines isolated for expression of a particular human bitter receptor T2R41 were cultured in individual wells of a 96-well plate and the presence of viable cells in each well was confirmed by preliminary calcium sensitive dye loading measurements. Wells with no or low dye loading were treated as blanks and are shown in black in fig. 14. The cells were then tested for their response to the addition of bitter extract by functional bitter receptors, read by receptor-mediated calcium mobilization and monitored by changes in dye fluorescence. Wells containing cells but showing less than 2-fold increase in signal above the initial fluorescence were considered negative and are shown in white in fig. 14, whereas wells with more than 2-fold increase in signal are shown in grey. In this plate, 57 clones (89%) out of 64 clones isolated for the expression of the bitter taste receptor gene showed significant functional bitter taste receptor responses.

Example 30 functional real-time imaging of bitter taste receptor response

Homogeneity is another important parameter in the generation of stable cell lines. We used functional real-time imaging of bitter taste receptor responses to determine homogeneity of cells isolated using the method described in example 26 compared to cells found by drug selection alone. Cells expressing bitter taste receptors and G protein were plated on 96-well poly D lysine-coated black clear plates (Becton Dickinson) 24 hours prior to assay. Used in assay buffer (130NaCl, 2mM CaCl, 1.2mM MgCl) ₂Cells were loaded with calcium sensitive fluorescent dye diluted in 5mM KCl, 10mM glucose, 10mM HEPES, pH 7.4). Cells were incubated for 1 hour, followed by the use of known activators of bitter taste receptors at appropriate concentrations. The calcium flux response of the cells was recorded using an AxioVert 200 EPI-fluorescence microscope (Zeiss) using appropriate filters for 3 minutes. Data were analyzed using metamorph6.3r7 software (Molecular Devices).

The isolated clonal cell lines were tested for the response of the cognate bitter taste receptor (fig. 15, top photograph), as indicated by the increase in intracellular free calcium due to bitter taste receptor activation. In cultures of similarly treated drug-selected cells, substantial heterogeneity in assay response was observed following application of the same activator (fig. 15, lower photograph). This result shows that the method of producing a bitter taste receptor-expressing cell line of the present invention (i.e., example 26) can select a population of cells that are genetically and functionally identical.

Example 31 consistency of functional response in cell lines expressing bitter taste receptors

Because the bitter taste receptor is a G protein-coupled receptor (GPCR), it is important to determine the G protein-coupled functional identity in different cell lines in order to produce meaningful comparisons of bitter taste responses between different bitter taste receptors in the presence of different compounds. The relative responses of different cell lines across a range of concentrations of G protein-coupled agonist should be consistent. To validate this, we tested the identity of the G α 15-mediated receptor response in dose-response curve experiments with different concentrations of isoproterenol, an agonist of the 3-adrenergic receptor endogenously expressed in the cells, for all 25 cell lines expressing different human bitter taste receptors and mouse G α 15 protein. This endogenous receptor, when stimulated, can couple to expressed G α 15 and induce changes in intracellular free calcium. The percent response was plotted as a function of isoproterenol concentration, and the results were curve-fitted to calculate EC ₅₀Values (concentration of half maximal receptor activation) (see figure 16). This EC was found by comparing clonal bitter cell lines spanning all 25 isolates₅₀The values were 4.9. + -. 0.41nM, closely corresponding to published literature values, and showed minimal variation between cell lines.

Example 32 identification of functionally responsive broadly regulated, moderately regulated and selective receptors

Cell lines stably expressing the native human bitter taste receptor gene generated using the method of the invention (i.e., example 26) were tested in a functional cell receptor-based study to detect their activation by a collection of chemically diverse compounds. The substances 1-12 that induce dose-dependent activation of the corresponding human bitter taste receptors are listed next to the receptors they were found to activate in FIG. 17. This allows the identification of broadly, less broadly, and narrowly regulated receptors.

Example 33 identification of receptors activated by Compound libraries

The compound library was tested for activity against each of the 25 human bitter taste receptor cell lines. Data regarding receptor activity after addition of compound was generated in a functional cell-based assay and expressed as a percentage activity above the baseline activity of the receptor (i.e., receptor activity when buffer alone was added). The activity of each compound at each receptor was then encoded as a highlighted compound with low (< 100% greater than baseline activity; white), medium (101-. The resulting pattern provides a graphical representation of compounds (rows) with selective or broad activity between bitter taste receptors and receptors (columns) that exhibit broad or selective responses to a chemically diverse group of compounds.

Example 34 has similar function to an antagonist of agonist-induced bitter taste receptor activity Identification of analogs of active compounds

To test the ability to identify analogs of compounds with similar functional activity based on assays of cells expressing bitter taste receptors, bitter taste receptors of 21 structural analogs of known bitter taste blocking compounds were tested for their ability to inhibit agonist-induced activity. The structures of analogs that inhibit the activity of bitter taste receptors induced by agonists were compared. Thus, cell lines expressing bitter taste receptors may aid in the discovery of potent bitter taste receptor antagonists.

Example 35 assignment of different receptors Using native and labeled cell lines

Functional assays performed after transient transfection as described in example 28 showed that native and labeled human bitter taste receptors have different functional activities. This was also verified using cell lines expressing the native bitter taste receptor in comparison to cell lines expressing the labeled bitter taste receptor. Briefly, bitter taste receptors to saccharin are generated using a functional cell-based receptor assay that measures calcium mobilization using a stable cell line expressing a native bitter taste receptorAssignment of value. Such as Pronin et al, "Identification of Ligands for Two Human Bitter cutter T2R Receptors", chem. Senses, 29: 583-593(2004), the use of insect-produced recombinant receptor proteins with the N-terminal mouse rhodopsin tag sequence to generate bitter receptors to saccharin in a membrane fraction-based cell-free assay for GTP hydrolysis And (7) assigning values.The different assignments are shown in fig. 19. The difference between bitter taste receptor assignments for the same bitter compounds highlights the natural bitter taste receptor as a prerequisite for physiologically relevant functional assignments.

Example 36 Generation of Stable sweet taste receptor expressing cell lines

Transfection

HEK293T (ATCC CRL-11268) was co-transfected with 3 separate plasmids, one encoding T1R2(SEQ ID NO: 31), one encoding T1R3(SEQ ID NO: 32) and the other encoding a signaling molecule (mouse G.alpha.15, SEQ ID NO: 33). Although drug selection is optional in the methods of the invention, we include a drug resistance marker/plasmid. The sequence is under the control of the CMV promoter. The untranslated sequence encoding the tag for detection by the signaling probe is also present along with the sequence encoding the drug resistance marker. The target sequences utilized were target sequence 1(SEQ ID NO: 28, using the target sequence of SEQ ID NO: 123), target sequence 2(SEQ ID NO: 29, using the target sequence of SEQ ID NO: 124) and target sequence 3(SEQ ID NO: 30, using the target sequence of SEQ ID NO: 125). In these examples, the T1R2 gene vector comprises target sequence 3, the T1R3 gene vector comprises target sequence 1, and the ga 15 gene vector comprises target sequence 2. Cells are routinely selected in media containing drugs for 10-14 days.

Contacting the cell with a fluorescent probe

Selected cells were harvested and transfected with the signaling probe (SEQ ID NOS: 38-40). Signaling probe 1 binds to tag sequence 1, signaling probe 2 binds to tag sequence 2 and signaling probe 3 binds to tag sequence 3. Cells were then dissociated and collected for analysis and sorted using a fluorescence activated cell sorter (Beckman Coulter, Miami, FL).

As will be appreciated by one of ordinary skill in the art, any agent suitable for use with the selected host cell can be used to introduce nucleic acids (e.g., plasmids, oligonucleotides, labeled oligonucleotides) into the host cell with appropriate optimization. Examples of reagents that can be used to introduce nucleic acids into a host cell include, but are not limited to, Lipofectamine 2000, Oligofectamine, TFX reagens, Fugene 6, DOTAP/DOPE, Metafectine, or F ecturin.

Signal transduction probe 1

5 '-Cy 5 GCCAGTCCCAGTTTCCTGTGTGCCTTAAGACCTCGC BHQ3 quenching-3' (SEQ ID NO: 38)

Signal transduction probe 2

5 '-Cy5.5GCGAGTCGCAGAACGACAGGGTTAACTTCCTCGCBHQ3 quenching-3' (SEQ ID NO: 39)

Signal transduction probe 3

5 '-Fam GCGAGAGCGACAAGCAGACCTATAGAACCTCGCCBHQ 1 quench-3' (SEQ ID NO: 40)

Target sequence 1 (sweet taste) with target sequence shown in bold

AGGGCGAATTCCAGCACACTGGCGGCCGTTACTAGTGGATCCGAGCTCGGAGGCAGGTGGACAGGAAGGTTCTAATATCTGGGCCCGGAAAGCCTTTTTCTCTGTGATCCGGTACAGTCCTTCTGC(SEQ ID NO：123)

Target sequence 2 (sweet) T with target sequence shown in bold

AGGGCGAATTCCAGCACACTGGCGGCCGTTACTAGTGGATCCGAGCTCGGTACCAAGCTTCGAGGCAGGTGGACAGCTTGGTTCTAATATCTGGGCCCGGAAAGCGTTTAACTGATGGATGGAACAGTCCTTCT GC (SEQ IDNO：124)

Target sequence 3 (sweet taste) with target sequence shown in bold

AAGGGCGAATTCGGATCCGCGGCCGCCTTAAGCTCGAGGCAGGTGGACAGGAAGGTTCTAATATCTGGGCCCGGAGATG(SEQ ID NO：125)

Other target sequences and signaling probes can be used (see, e.g., International patent application publication No. WO2005/079462 (application No. PCT/US 05/005080)) as published on 9/1/2005. For example, BHQ3 may be replaced by BHQl or gold particles in probe 1 or probe 2. It should be noted that BHQ1 can be replaced by BHQ2 or Dabcyl in Probe 3. Similar probes using Quasar Dye (BioSearch) with similar spectral properties as Cy5 can be used in certain experiments. It should also be noted that in some cases 5-MedC and 2-aminodA mixmer probes are used instead of DNA probes.

Positive forIsolation of cells

Standard assay methods are used to gate cells that fluoresce above background and isolate individual cells that fall within the gate into 96-well plates. Cell sorting was operated so that a single cell was placed per well. After selection, the cells are expanded in medium in the absence of the drug.

Functional transformation

We maintained sweet taste receptor cells (selected and expanded as above) using aliquots of the same cells and different cells in a variety of growth conditions, including, for example, low glucose DMEM media or in glucose-free Leibovitz L-15 media with 10% serum or serum-free. Some cells were maintained in media containing different concentrations of other sugars, such as galactose. We subsequently characterized cells of the sweet taste receptor cell line maintained under various conditions by their ability to respond appropriately to sweet taste ligands and not to respond to other stimuli (MSG).

Without wishing to be bound by theory, growth in different media conditions may functionally transform cells, for example, due to changes in gene expression levels, genomic tissue, and functional expression of receptors on the cell surface. Parameters in different media evaluated and shown to affect the functional assay response of cells include serum concentration (i.e., low serum concentration and serum deprivation), sugar deprivation, and assay plate coating (e.g., poly-D-lysine and laminin). These results show that the characteristics of the cells of the sweet taste receptor cell line can vary when the cells are maintained in different media conditions. They also show that different cells selected by fluorescent probes can have different characteristics under the same growth conditions. An example of our characterization of cells selected by fluorescent probes is shown in figure 20. As shown in figure 20, aliquots of the same cells cultured in different conditions (1, 2 and final) responded significantly differently to umami (MSG) and sweet (fructose) ligands. The cells cultured in condition 1 were plated in a low glucose medium deprived of serum. The cells cultured in condition 2 were plated overnight in low glucose medium containing 10% serum and then switched to serum deprived medium before measurement. Cells cultured in condition 1 or 2 were plated on a coated plate (Corning # 3665). "Final" conditions included high density cultures on coated plates (Corning #3300) and low glucose media with serum deprivation. Cells cultured in conditions 1 and 2 respond to umami agonist more than to sweet agonist. In contrast, cells cultured in "final" culture conditions responded strongly to fructose but not to MSG. Thus, these cells produce physiologically and pharmacologically relevant sweet taste receptors.

Example 37 Generation of cell lines based on Natural sweet taste receptor function

1. Verification and quantification of gene expression

By using qRT-PCR, we determined the relative amount (RNA) of each umami receptor subunit expressed in the above cells ("final"). From 1-3X 10 using a commercially available RNA purification Kit (RNeasy Mini Kit, Qiagen)⁶Total RNA was purified from individual mammalian cells. The RNA extract was then treated using a stringent DNase treatment protocol (TURBO DNA-free Kit; Ambion). First strand cDNA synthesis was performed in 20. mu.L using the Reverse transcription kit (Superscript III, Invitrogen) using 1. mu.G total RNA without DNA and 250nG random primers (Invitrogen). Negative controls for this reaction include samples in which reverse transcriptase or RNA is not added during the cDNA synthesis step. cDNA and PCR product synthesis was performed in a thermal cycler (Mastercycler Eppendorf) under the following conditions: at 25 ℃ for 5 minutes and at 50 ℃ for 60 minutes; the reaction was terminated at 70 ℃ for 15 minutes.

To analyze gene expression (RNA), probes for T1R2, T1R3, and mouse G.alpha.15 cDNA (MGB TaqMan probes, Applied Biosystems) were used. For the sample normalization control GAPDH, use was made of Pre-Developed TaqMaN Assay reagent, applied biosystems. Reactions were set up in triplicate in a 50 μ L reaction volume using 40ng of cDNA, including negative and positive controls (plasmid DNA). The relative amount of each sweet taste receptor subunit expressed (RNA) was determined. Figure 21 graphically depicts the results and shows that all 3 nucleic acids are expressed (RNA) in sweet taste receptor cell lines. Expression levels of T1R2, T1R3, and ga 15 were approximately 100,000, 10, and 100,000 times the levels observed in control cells, respectively.

Standard single-endpoint RT-PCR methods were used to estimate gene expression (RNA) of T1R2, T1R3, and ga 15 in 1 and 9 month cultures of sweet taste receptor cell lines generated according to the protocol described above ("final"). Cells were cultured in a 24-well plate format to 80% confluence, harvested and RNA isolated using a commercially available RNA preparation kit (RNAqueous kit, Ambion). Purified total RNA in the range of 5pg to 5. mu.g was used for reverse transcription using oligo (dT)12, 18 primers according to the protocol of a commercial first strand cDNA synthesis kit (Superscript III kit, Invitrogen). Following first strand synthesis, nucleic acids specific for subunits of sweet taste receptors (T1R2, T1R3) and the nucleic acid group of mouse G α 15 were independently assembled in a PCR reaction mixture (HotStart Taq). After 45 cycles of PCR, the amplicon samples were further analyzed using agarose gel electrophoresis.

The results from these single-endpoint RT-PCR experiments are illustrated in figure 22. FIG. 22 depicts representative photographs of agarose gels used for RT-PCR experiments. The intense expression (RNA) of all nucleic acids encoding sweet taste receptors was detected in cultures of 1 month and longer, 9 months, demonstrating an abnormal level of stability of the cell lines of the invention cultured under "final" conditions.

2. Cell-based assays for modulators

Cells of the invention were seeded at high density in growth medium (low glucose DMEM or L15 medium supplemented with serum and standard growth additives) in 10cm dishes 48 hours before the assay. Cells were dissociated from the culture matrix and seeded in 96-well plates 24 hours prior to assay. The medium was removed and then calcium sensitive fluorescent dye (calcium-3, Molecular devil) was addedces Corp.), the dye was diluted in sweet assay buffer (130mM NaCl, 1.1mM KH)₂PO₄，1.3mM CaCl₂20mM HEPES and 3mM NaHPO₄*7H₂O). Cells were incubated in this medium for 1 hour. The plates were then loaded on a high throughput fluorescence plate reader (Hamamatsu FDSS). Test compounds (i.e., fructose (Sigma), sucrose (Sigma), glucose (Sigma), acesulfame k (fluka), sodium saccharin (Sigma), sodium cyclamate (Aldrich), stevia (Steviva brands inc.), mogroside (Slim Sweet, Trimedica), and sorbitol (Sigma)) were diluted in Sweet assay buffer and added to each well. Calcium flux was measured for 90 seconds. Activator (i.e., MSG) diluted in the above buffer was added to each well at a final concentration ranging between 10. mu.M to 100mM, and the change in relative fluorescence was recorded for an additional 90 seconds.

3. Determination of Z' and EC according to sweet cell-based assays ₅₀ Value of

To test the efficacy of sweet taste receptor responses in such sweet taste receptor expressing cells, the established sweet taste receptor activator fructose was used as a test compound in the above assay. Fructose was added to test wells (odd columns) at a concentration of 75mM and control wells (even columns) received buffer only. In the final assay conditions as reported by calcium flux measurements measured by fluorescent plate readers (FLIPR3 operating system, Molecular Devices). In one assay repeated several times, the cells had a Z' value of 0.8. See fig. 23 (an exemplary assay). The Z' value indicates that the resulting cells expressing sweet taste receptors recognize fructose in cell-based assays, and that such assays can be reliably and stably performed using such cells.

To test the sensitivity of sweet taste receptor responses in the sweet taste receptor expressing cell lines of the invention, a series of dose response experiments were performed by adding increasing doses of various sweet taste receptor agonists to the cells and then measuring the responses as described above. In one assay (figure 24),finding the EC of such test Compounds₅₀The values are as follows: less than 1.5mM for saccharin, less than 3.5mM for sucrose, less than 4.3mM for fructose and less than 34.1mM for glucose. These results indicate that the sweet taste receptor produced in the cell line of the invention exhibits strong sensitivity to many known sweet taste receptor ligands in the cell line of the invention expressing sweet taste receptors.

In this assay, it was also found that the calcium flux response curves were different when cells expressing sweet taste receptors were contacted with different sweeteners. As illustrated in fig. 25, the length and intensity of the response produced by the cells to different ligands can vary. For example, stevia showed a longer and stronger response than many of the other sweeteners tested. These results suggest that the cells and cell lines of the invention are useful for the determination of the sweetness delay (desired persistence of sweetness) seen with certain high intensity sweeteners, such as stevia.

Example 38 Stable cell lines endogenously expressing at least one sweet taste receptor subunit Generation and separation

Design of signaling probes

Signaling probes are designed to the sequences corresponding to the T1R2 or T1R3 loci. The signaling probes are directed to coding exons, non-coding introns, or non-coding untranslated sequences listed in table 23.

The signaling probes S21, S22, S23, R2-3U1, and R2-I1 comprise a Cy5.5 fluorescent label at the 5 'end and a BHQ2 quencher at the 3' end. The signaling probes S31, S32, S33, R3-3U1, and R3-I31 contained a 6-FAM fluorophore at the 5 'end and BHQ1 at the 3' end. The signaling probe may also comprise other fluorescent groups or quenchers. Signaling probes were synthesized, conjugated to their respective labels and purified in Genelink (Hawthorne, NY).

TABLE 23

FIGS. 26 and 27 depict graphical representations of T1R2 and T1R3 loci and intron-exon coding structures. Other target sequences and signaling probes can be used (see, e.g., International patent application publication No. WO2005/079462 (application No. PCT/US 05/005080)) as published on 9/1/2005. For example, BHQ1 can be replaced by BHQ2 or Dabcyl in probe 3. It should also be noted that in some cases 5-MedC and 2-aminodA mixmer probes may be used instead of DNA probes.

Contacting the cell with a fluorescent probe

HEK293T (ATCC CRL-11268), HuTu (ATCC HTB-40) or H716(ATCCCCL-251) cells were transfected with one of the following combinations of signaling probes in separate experiments: a) s21 and S31, b) S21 and S32, c) S21 and S33, d) S22 and S31, e) S22 and S32, f) S22 and S33, g) S23 and S31, h) S23 and S32, I) S23 and S33, j) R2-3U1 and R3-3U1, k) R2-3U1 and R3-I31, l) R2-I1 and R3-3U1 and m) R2-I1 and R3-I31. HEK293T was maintained in Darbeike ' S modified eagle ' S medium (Sigma # D5796) containing 10% Fetal Bovine Serum (FBS) (Sigma # SAC1203C), 2mM L-glutamine (Sigma G7513) and 10mM HEPES (Sigma H0887), HuTu cells were maintained in eagle ' S basal fraction medium (Sigma # D5650) containing 10% FBS (Sigma #2442), 2mM L-glutamine (Sigma G7513) and 1mM sodium pyruvate (Sigma # S8636) and H716 cells were maintained in Roswell Park medical Institute 1640(Gi # 11875-. As will be appreciated by one of ordinary skill in the art, any agent suitable for use with the selected host cell can be used to introduce nucleic acids (e.g., plasmids, oligonucleotides, labeled oligonucleotides) into the host cell with appropriate optimization. Examples of reagents that can be used to introduce nucleic acids into a host cell include, but are not limited to, Lipofectamine 2000, Oligofectamine, TFX reagens, Fugene 6, DOTAP/DOPE, Metafectine, or F ecturin.

In various experiments, HEK 293T cells, HuTu cells and H716 cells were treated with or without mutagenic treatment (e.g., ultraviolet light) to induce random mutagenesis or gene activation prior to introduction of fluorescent probes as described below. Cells were plated at 1.25X 10⁶The individual cells/ml were resuspended in serum-free medium and then exposed to ultraviolet radiation to deliver 1 Joule/m from a germicidal bulb (Sylvania) emitting predominantly 254nM wavelength²Per second, for 15 minutes. Other mutagenic treatments (e.g., chemical mutagenesis by contact with a mutagen such as Ethyl Methane Sulfonate (EMS)) can also be used. The cells may be incubated with a final concentration (e.g., 200. mu.g/ml) of ethyl methanesulfonate (EMS; Sigma) and incubated before they are transferred to a medium in the absence of EMS.

The cells were then contacted with signaling probes, dissociated, and collected using a fluorescence activated cell sorter (Beckman Coulter, Miami, FL) for testing.

Isolation of Positive cells

The positive fluorescing cells are gated or selected according to standard practice in the field of flow cytometry and standard analytical methods familiar to those of ordinary skill in the art. Fluorescence activated cell sorting was used to isolate positively fluorescing cells that were classified within the desired gates or have the desired fluorescence directly into 96-well plates. Cell sorting parameters were set for the separation of one cell per well placement. Transfected cells associated with different absolute or relative fluorescence levels of at least one signaling probe were isolated by gating 1% of the cells with the highest fluorescence intensity relative to the entire population at the tip.

In separate experiments, transfected cells associated with different absolute or relative fluorescence levels of at least one signaling probe were isolated by gating 0.1% of the cells with the highest signal intensity relative to the whole population at the tip.

Individual cells were isolated and maintained in culture as described below. For each cell type, an equal proportion of its respective conditioned medium and fresh medium was used in combination. Conditioned media was prepared by culturing each cell type in its respective fresh medium for 2 days, harvesting, and then filtering the medium. Individually isolated HEK 293T cells were isolated and maintained in darbeke modified eagle medium (Sigma # D5796) containing 10% FB S (Sigma # SAC 1203C), 2mM L-glutamine (Sigma G7513), 10mM HEPES (Sigma H0887), and a 1: 50 diluted penicillin/streptomycin solution (final concentration of 100 units penicillin/100 μ G streptomycin/mL) (Sigma # P4458). Individually isolated HuTu cells were isolated and maintained in eagle' S basal medium (Sigma # D5650) in 10% FBS (Sigma #2442), 2 mL-glutamine (SigmaG7513), 1mM sodium pyruvate (Sigma # S8636), and a 1: 50 diluted penicillin/streptomycin solution (final concentration of 100 units penicillin/100 μ g streptomycin/mL) (Sigma # P4458). Individual isolated H716 cells were isolated and placed in a Roswell Park molar Institute1640(Gibco #11875-093) containing 10% FBS (Sigma # F2442) and a 1: 50 diluted penicillin/streptomycin solution (final concentration of 100 units penicillin/100. mu.g streptomycin/mL) (Sigma # P4458).

The isolated cells were passaged using a robotic cell culture method and maintained in 96-well tissue culture plates. Specifically, MICROLAB STAR^TMThe instrument is used to perform many automated cell culture tasks (e.g., removing culture medium, replacing culture medium, adding reagents, cell washing, removing wash solution, adding dispersants, removing cells from culture vessels, adding cells to culture vessels, etc.). Other cell culture methods, including manual methods, may also be used. The cells are then expanded in 10cm tissue culture dishes and cultured using standard manual tissue culture techniques (e.g., media removal, cell washing, media addition).

The isolated cells are expanded in culture to produce a population of cells derived from the individually isolated cells. The individually isolated cells were expanded by culturing them in their respective media as described below. HEK 293T cells were transferred to be maintained in darbeke modified eagle medium (Sigma # D5796) containing 10% Fetal Bovine Serum (FBS) (Sigma # SAC1203C), 2mM L-glutamine (SigmaG7513) and 10mM hepes (Sigma H0887), and HuTu cells were transferred to be maintained in eagle' S basal component medium (Sigma # D5650) containing 10% FBS (Sigma #2442), 2mM L-glutamine (SigmaG7513) and 1mM sodium pyruvate (Sigma # S8636). Transfer H716 cells were maintained in a Roswell Park Memorial Institute 1640(Gibco #11875-093) containing 10% FB S (Sigma # F2442).

Functional testing

The populations of cells each resulting from the expansion of a single isolated cell are then characterized in terms of their ability to respond to the sweet taste ligand using a functional cell-based assay. 15mM fructose was used in these experiments. Depending on the cell type, different conditions were used as described below.

Cells were seeded at a high density of 1200 to 2000 million cells in growth medium L15 medium (Sigma # L5520) supplemented with 10% serum (Sigma # SAC1203C), 4 mML-glutamine (Sigma G7513), 10mM HEPES (Sigma H0887), and a 1: 50 dilution of penicillin/streptomycin solution (final concentration of 100 units penicillin/100. mu.g streptomycin/mL) (Sigma # P4458) in 10cm dishes 48 hours prior to the assay. Cells were dissociated from the culture matrix and seeded 24 hours prior to assay in 384-well plates at 35,000 cells/well. The medium was removed after 18 to 24 hours. Subsequently, each well was diluted in a sweetness assay buffer (130mM NaCl, 1.1mM KH)₂PO₄，1.3mM CaCl₂20mM HEPES and 3mM NaHPO₄*7H₂O) calcium-sensitive fluorescent dye (Fluo-4, Invitrogen). Cells were incubated in this medium for 1 hour. The plates were then loaded on a high throughput fluorescence plate reader (Hamamatsu FDSS). Add sweetness assay buffer and DMSO to 0.2% DMSO in each well Final concentration. Calcium flux or assay reactions were detected before, during and after adding sweet assay buffer containing fructose to a final concentration of 15mM fructose to each well. The assay can optionally be performed with quenchers for calcium sensitive fluorescent dyes. Such quenchers are well known in the art and include, for example, bispicramine (DPA), acid Violet 17(AV17), Diazine Black (Diazine Black) (DB), HLB30818, Trypan blue, bromophenol blue, HLB30701, HLB30702, HLB30703, nitrazine yellow, nitrored, DABCYL (molecular probes), QSY (molecular probes), metal ion quenchers (e.g., Co, etc.)²⁺、Ni²⁺、Cu²⁺) And iodide ions.

Alternatively, cells were dissociated from the culture medium 24 or 48 hours prior to the assay, and seeded in 384-well plates at a cell density of 2,500-. The medium was removed after 18 to 52 hours. Subsequently, each well was diluted in a sweetness assay buffer (130mM NaCl, 1.1mM KH) ₂PO₄，1.3mM CaCl₂20mM HEPES and 3mM NaHPO₄*7H₂O) calcium-sensitive fluorescent dye (Fluo-4, Invitrogen). Cells were incubated in this medium for 1 hour. The plates were then loaded on a high throughput fluorescence plate reader (Hamamatsu FDSS). Sweetness assay buffer and DMSO were added to each well to a final concentration of 0.2% DMSO. Calcium flux or assay reactions were detected before, during and after adding sweet assay buffer containing fructose to a final concentration of 15mM fructose to each well.

Alternatively, the cells are pelleted and then resuspended in diluted sweetness assay buffer (130mM NaCl, 1.1mM KH)₂PO₄，1.3mM CaCl₂20mM HEPES and 3mM NaHPO₄*7H₂O) in a calcium-sensitive fluorescent dye (Fluo-4, Invitrogen). Cells were plated in 384-well plates and incubated in this medium for 1 hour. The plate is then loaded highFlux fluorescence plate reader (Hamamatsu FDSS). Sweetness assay buffer and DMSO were added to each well to a final concentration of 0.2% DMSO. Calcium flux or assay reactions were detected before, during and after adding sweet assay buffer containing fructose to a final concentration of 15mM fructose to each well.

Other Sweet taste acceptors (including high intensity natural and artificial sweeteners and natural and artificial sweeteners) may also be used, such as sucrose (Sigma), glucose (Sigma), acesulfame k (fluka), sodium saccharin (Sigma), sodium cyclamate (Aldrich), Steviva (Steviva brands inc.), mogroside (Slim Sweet, Trimedica) and sorbitol (Sigma). In addition, other taste ligands including bitter, umami, fatty, refreshing, pungent and sour ligands or tastants as well as compounds with unknown or inherent taste may be used to determine the selectivity and specificity of the sweet taste response. In addition, any tastant or ligand may be tested in the presence of other compounds, including enhancers or blockers, such as the sweetness enhancer rebaudioside-C RP44(RedPoint Bio), as well as the sucralose enhancer S2383 (senomoyx) and the sucrose enhancer S5742 (senomoyx).

In addition, other media and incubation steps can be used as well as other plate formats such as 96-well plates and 1536-well plates can be used.

By monitoring the response or activation kinetics of cells in a functional fluorescent calcium flux reporter assay, a number of cell populations cultured from individually isolated cells and found to express at least one subunit of a sweet taste receptor, either naturally and/or by gene activation, are identified as having a response to fructose. See, fig. 8. Fig. 28A shows HuTu cells responding to fructose. Fig. 28B shows H716 cells responding to fructose. Figure 28C shows 293T cells responding to fructose.

High throughput screening

Sweet taste agonists and/or modulators may be identified by testing the effect of compounds on cells plated according to the methods described above using High Throughput Screening (HTS). A sweet assay buffer is added to each well with or without one or more components or compounds of a natural or synthetic compound library or extract fraction. DMSO or other organic solvents such as ethanol are also used to help solubilize the compound or extract to a final concentration of less than 5% DMSO (e.g., 0.2%). The detection of calcium flow is performed for a period of time (e.g., 90 seconds). Fructose or another sweet ligand diluted in sweet assay buffer is then added to each well to a final concentration (e.g., 15 mM). The change in relative fluorescence is recorded for another period of time (e.g., 90 seconds). A number of control compounds will also be tested.

This test is repeated until some or all of the compound library has been screened. The data are analyzed to identify the active compound. Agonists and modulators, including blockers, enhancers or allosteric modulators, are identified using this or other assay protocols. Testing of compounds using different sweet taste ligands is used to identify compounds that modulate the response of sweet taste receptors to only one, all or a subset of sweet taste ligands, e.g., compounds that are selective or pan-active. This is done by independent high throughput screening of the same compound or compound library, but using one or more different sweet ligands in each screen.

By examining the time kinetics of the reaction curve (e.g., the decay or magnitude or rate of reaction), regulatory compounds with other qualitative and quantitative effects can be identified.

Taste test

Compounds identified using sweet cell-based assays are tested in human sensory testing according to standard practice in the art and familiar to those of ordinary skill in the art. The inherent taste characteristics of the test compounds (e.g., sweetness, bitterness, umami, sourness, refreshing or pungent taste, and aftertaste) as well as other characteristics or properties that can be perceived such as mouthfeel, numbing, pain, irritation, and tingling sensation. Samples with and without compound were evaluated to detect the sensory properties of the compound. The test is carried out in one or more different forms or matrices, including liquid, semisolid, solid, powder, tablet, capsule, jelly, spray, dissolvable strip, food or beverage formulation. Samples with or without a compound are evaluated to detect the sensory properties of the compound. Different doses of the compound can be tested. In certain experiments, compounds are tested in samples that additionally contain other compounds or components with a taste, and the change, alteration, increase, enhancement, or inhibition of the perception of such taste is assessed. The compound may be provided in the presence of an agent designed to dissolve it, including an alcohol such as ethanol, DMSO, or other solvent. The sample may be heated, frozen or tested at room temperature. Compounds may also be tested for their ability to enhance or block sweetness or other taste. Sensory testing was performed with or without a nose clip to identify any odor-related effects. Taste tests were performed using a "sniff" mode, in which low concentrations of the compounds were used to limit human exposure. Tasting compounds to determine their effect on the perception of one or more sweet taste ligands and sweeteners. A human or animal subject contacted with or assessed for a compound can be studied using functional brain imaging or other imaging assays (e.g., magnetic resonance imaging) to correlate a pattern of activity in vivo with the compound.

Example 39 Generation of Stable odorant receptor expressing cell lines

Step 1 transfection

293T cells were co-transfected with 2 separate plasmids, one encoding human odorant receptor (one of SEQ ID NO: 134-135) and the other encoding human G.alpha.15 signaling protein (SEQ ID NO: 133). As will be appreciated by one of ordinary skill in the art, any agent suitable for use with the selected host cell can be used to introduce nucleic acids (e.g., plasmids, oligonucleotides, labeled oligonucleotides) into the host cell with appropriate optimization. Examples of reagents that can be used to introduce nucleic acids into a host cell include, but are not limited to, Lipofectamine 2000, Oligofectamine, TFX reagent, gene6, DOTAP/DOPE, Metafectine, or Fecturin. Although drug selection the methods described herein are optional, we include a mammalian drug-resistance marker/plasmid. The plasmid used herein contains the CMV promoter for expression of the odorant receptor gene or the ga 15 gene. An untranslated sequence encoding a tag (which comprises the target sequence) for detection by a signaling probe is present in each plastid, such that the tag is either co-transcribed with or fused to the odorant receptor or the G α 15 transcript. The tag comprises a target sequence capable of hybridizing to the signaling probe. The target sequences utilized were target sequence 1(SEQ ID NO: 129) and target sequence 2(SEQ ID NO: 130), and the target sequences utilized were target sequence 1(SEQ ID NO: 136) and target sequence 2(SEQ ID NO: 137). In these examples, the plasmid encoding the odorant gene comprises target sequence 1 and the plasmid encoding the ga 15 gene comprises target sequence 2.

Step 2: selection step

The transfected cells were cultured in Darbevaceae Modified Eagle Medium (DMEM) containing Fetal Bovine Serum (FBS) for 2 days, followed by culture in DMEM-FBS containing antibiotics for 2 weeks. The antibiotic-containing period had the following antibiotics added to the medium: puromycin (0.3. mu.g/ml) and hygromycin (110. mu.g/ml), puromycin (0.15. mu.g/ml) and hygromycin (75. mu.g/ml) and puromycin (0.05. mu.g/ml) and hygromycin (18. mu.g/ml).

And step 3: cell passage

After enrichment by antibiotic selection, and prior to introduction of the fluorescent signaling probe, cells were passaged 3 to 15(p3-p15) times or more in the absence of antibiotic to allow time for any unstable expression to subside.

And 4, step 4: contacting a cell with a fluorescent signaling probe

Cells were harvested and transfected with signaling probes (SEQ ID NOS: 131 and 132). As will be appreciated by one of ordinary skill in the art, any agent suitable for use with the selected host cell can be used to introduce nucleic acids (e.g., plasmids, oligonucleotides, labeled oligonucleotides) into the host cell with appropriate optimization. Examples of reagents that can be used to introduce nucleic acids into a host cell include, but are not limited to, Lipofectamine 2000, Oligofectamine, TFX reagens, Fugene 6, DOTAP/DOPE, Metafectine, or Fecturin.

Target sequence detection by a signaling probe

Target sequence 1

5′-GTTCTTAAGGCACAGGAACTGGGAC-3′(SEQ ID NO：129)

Target sequence 1, target sequence in bold (odor)

5′-AGGGCGAATTCCAGCACACTGGCGGCCGTTACTAGTGGATCCGAGCTCGGAGGCAGGTGGACAGGAAGGTTCTAATCATCTGGGCCCGGAAAGCCTTTTTCTCTGTGATCCGGTACAGTCCTTCTGC-3′(SEQ ID NO：136)

Target sequence 2

5′-GAAGTTAACCCTGTCGTTCTGCGAC-3′(SEQ ID NO：130)

Target sequence 2, target sequence in bold (odor)

5′-AGGGCGAATTCCAGCACACTGGCGGCCGTTACTAGTGGATCCGAGCTCGGTACCAAGCTTCGAGGCAGGTGGACAGCTTGGTTCTAATATCTGGGCCCGGAAAGCGTTTAACTGATGGATGGAACAGTCCTTCTGC-3′(SEQ IDNO：137)

Signal conduction probe

The signaling probe was supplied as a 100. mu.M stock solution.

Signaling probe 1-binding target 1

5 '-Quasar 670 GCCAGTCCCAGTTTCCTGTGTGCCTTAAGAACCTCGCCBHQ 2 quenching-3' (SEQ ID NO: 131)

Signaling probe 2-binding target 2

5 '-Cy5.5GCGAGTCGCAGAACGACAGGGTTAACTTCCTCGCBHQ 2 quenching-3' (SEQ ID NO: 132)

It should also be noted that in some cases 5-MedC and 2-aminodA mixmer probes are used instead of DNA probes.

Random non-targeted 6-fam probes were used as delivery controls in some experiments (not shown).

And 5: isolation of Positive cells

Cells were dissociated and collected for analysis and sorted using a fluorescence activated cell sorter (Beckman Coulter, Miami, FL). Standard assay methods are used to gate cells that fluoresce above background and isolate individual cells that fall within the gate into a barcoded 96-well plate. The gating levels are as follows: coincidence gate > unimodal gate > valve > sorting gate. With this gating strategy, the top 0.1-2% of double positive cells were marked for sorting into barcoded 96-well plates.

Step 6: additional cycles of Steps 2-5 and/or 3-5

The experiment was staged with very compact timing logic. As part of this protocol, two full cycles of steps 2-5 or steps 3-5 are performed to complete redundant sorting and obtain additional clones.

Step 7-estimation of growth Rate of cell population

The plates were transferred to a Hamilton Microlabstar automated liquid handler. Cells were grown on 2-3 day conditions in medium: incubation in a 1: 1 mixture of fresh growth medium (DMEM/10% FBS) supplemented with 100 units penicillin/ml plus 0.1mg/ml streptomycin for up to 20 days. Plates were imaged 7 to 30 days after sorting to determine well confluence (Genetix). Each plate is focused for reliable image acquisition across the plate. Not relying on reported confluencies of more than 70%. Confluent measurements were obtained 3 times over the time specified above and used to calculate growth rate.

Step 8-estimating the Box and cell population according to growth Rate

The cells were boxed (grouped independently and plated as a cohort) according to a growth rate of about 20-30 days after sorting. Frames are collected independently and plated on individual 96-well plates for downstream processing, and there may be more than one target plate/specific frame. The boxes were calculated by considering the spread in growth rate and classifying ranges covering a high percentage (at least about 30-90% (as an estimate)) of the total number of cell populations as homogeneous. In the across different odor taste receptor cell lines in the frame using about 15-40 hours average separation of 9 growth frame. Each box thus corresponds to a difference in growth rate or population doubling time between about 1 and 5 hours.

Cells may have doubling times of less than 1 day to more than 2 weeks-in order to process the most diverse clones that can be simultaneously binned reasonably according to growth rate; we generally prefer to generate 3-9 boxes with a doubling time of 0.25-0.7 days per box. One of ordinary skill in the art would understand how to adjust the tightness of the boxes and the number of boxes for a particular situation, and further adjust the tightness and number of boxes if the cells are synchronized according to their cell cycle. In this example, cell lines with doubling times of 15 to 40 hours were selected for framing and to increase the compactness of the frame.

The plates were subjected to standard and fixed conditions (humidified 37 ℃, 5% CO)₂95% air) in DMEM medium/10% FBS containing antibiotics (100 units penicillin/ml plus 0.1mg/ml streptomycin). The plates of cells were separated to generate 4 sets (a set consisting of all plates with all growth frames, these steps ensuring that there were 4 replicates of the starting set) of target plates. Up to 3 target plate sets were submitted for cryopreservation (see below)) And the remaining groups were scaled and further spread repeated for passage and for functional assays. Different and independent tissue culture reagents, incubators, personnel and carbon dioxide sources were used for each set of plates. Quality control steps were taken to ensure proper production and quality of all tissue culture reagents: each component added to each bottle of media prepared for use was added by one designated person in one designated fume hood, where only that reagent was present, while the second designated person was supervising to avoid errors. Conditions for liquid handling are set to eliminate cross-contamination between wells. Fresh tips were used for all steps, or a stringent tip washing protocol was used. Liquid handling conditions were set for accurate volume transfer, efficient cell treatment, wash cycles, pipetting speed and positioning, number of pipetting cycles for cell dispersion, and relative position of tip to plate.

Step 10: early passage stock of frozen cell populations

Groups 2 plates were frozen at-70 to-80 ℃. Plates in each group were first allowed to reach 70 to 100% confluence. The medium was aspirated and 90% FBS and 10% DMSO were added. Use of flat plateSealed and then surrounded with 1 to 5cm of foam, respectively, and placed into a-80 ℃ freezer.

The remaining 2 sets of plates were maintained as described in step 9. All cells were performed separately using automated liquid processing steps including media removal, cell washing, trypsin addition and incubation, quenching and cell dispersion steps.

Step 12: testing of functionality of cells in a rearranged plate

The consistency and standardization of cells and culture conditions for all cell populations were controlled. The difference across the plate due to slight differences in growth rate was controlled by normalization of the number of cells across the plate and was generated every 2-8 passages after rearrangement. Cell populations that are outliers are detected and eliminated.

Step 13: characterization of cell populations

Step 14: assessment of potential functionality of cell populations

The cell population is tested using functional criteria. The calcium mobilization dye Fluo-4(Invitrogen) was used according to the manufacturer's instructions. The HEK293T cell line stably expressing h-OR was maintained in DMEM medium supplemented with 10% fetal bovine serum and glutamine under standard cell culture conditions. One day prior to the assay, cells were harvested from stock solution plates and plated in 384-well assay plates with a black, transparent bottom. Assay plates were incubated at 5% CO₂Maintained in a 37 ℃ cell culture incubator for 22-48 hours. The medium was then removed from the assay plate and diluted in buffer (130mM NaCl, 5mM KCl, 2mM CaCl) was added₂、1.2mM MgCl₂10mM HEPES, 10mM glucose, pH 7.0) with calcium mobilization dye (Fluo-4, Invitrogen, Carlsbad, Calif.) diluted in the above buffer (130mM NaCl, 5mM KCl, 2mM CaCl)₂、1.2mM MgCl₂10mM HEPES, 10mM glucose, pH 7.0) as a quencher were incubated together at 37 ℃ for 1 hour. The quenchers identified above may be determined by quenchers well known in the art such as, for example, bispicramine (DPA), acid Violet 17(AV17), Diazine Black (DB), HLB30818. Bromophenol blue, HLB30701, HLB30702, HLB30703, nitrazine yellow, nitro red, DABCYL (molecular probes), QSY (molecular probes), and metal ion quenchers (e.g., Co) ²⁺、Ni²⁺、Cu²⁺) And iodide ion displacement. The assay plate was then loaded on a fluorescent plate reader (Hamamatsu FDSS) and added dissolved in the above buffer (130mM NaCl, 5mM KCl, 2mM CaCl)₂、，1.2mM MgCl₂10mM HEPES, 10mM glucose, pH 7.0) (4.5 mM heliotropin (Helinal) for OR3A1, 0.3mM Boraginal (Bourgeonal) for OR1D 2-FIG. 29A, 29B). Cells were tested in 384-well plates at various densities and analyzed for response. Multiple time points after coating are used, for example 12-48 hours after coating. Differences in assay response for different coating densities were also tested.

Figure 29A depicts a representative trace of functional cell-based response of cells expressing the human odorant receptor OR3a1 to heliotropin (to a final concentration of 4.5 mM) compared to DMSO vehicle background signal as a control. Test and control traces were overlaid. The results of the cell-based assay designed to report calcium flux using a fluorescent calcium signaling dye provided in fig. 29A indicate that the cells exhibit a response to heliotropin above background.

Figure 29B depicts a representative trace of functional cell-based response of cells expressing human odorant receptor OR1D2 to bociclellal (to a final concentration of 0.3 mM) compared to DMSO vehicle background signal as a control. Test and control traces were overlaid. The results of the cell-based assay designed to report calcium flux using fluorescent calcium signaling dye provided in fig. 29B indicate that the cells show a response to boehringer aldehyde above background.

Functional responses from experiments performed at low and higher passage numbers were compared to identify cells with the most consistent response over a defined period of 6-40 weeks after sorting. It is also noted that other cellular characteristics that change over time, such as the time it takes for a cell to detach followed by reattachment, are noted.

Step 16-further evaluation of cells

Cell populations that meet functional and other criteria are further evaluated to determine those most amenable to the production of viable, stable, and functional cell lines. The selected cell population is expanded in larger tissue culture dishes and the characterization steps described above are continued or repeated under these conditions. In this regard, additional standardization steps were introduced for consistent and reliable passaging. These include different plated cell densities, plate coating, passage times, culture dish size/form and coating, fluidics optimization, cell dissociation optimization (e.g., dissociation in the presence of trypsin in a cell dissociation buffer (Invitrogen), volume of dissociation reagents used and length of time of dissociation), and washing steps. In addition, cell viability at each passage was determined. Increasing manual intervention and more closely observing and monitoring the cells. This information is used to help identify and select the final cell line that retains the desired properties. Final and backup cell lines showing consistent growth, consistent (i.e., no morphological changes) proper attachment, and functional response were selected.

And step 17: establishment of cell banks

Step 18-testing of cell banks

Example 40 characterization of cell lines for Natural odorant receptor function

To identify and measure ligand-induced responses of HEK293T cells expressing odorant receptors, receptor activation was monitored by measuring changes in intracellular calcium levels triggered by the receptors. Cells were cultured overnight in standard growth medium in black clear bottom plates or as assay buffer (130mM NaCl, 5mM KCl, 2mM CaCl) ₂、1.2mM MgCl₂10mM HEPES, 10mM glucose, pH 7.0) was added to a black clear bottom plate. Cells were incubated for 1 hour at room temperature with the wash-free calcium sensitive fluorescent dye calcium-3 (molecular devices Corp.) in a buffer with as a quencher and diluted in the above buffer (130mM NaCl, 5mM KCl, 2mM CaCl)₂，1.2mM MgCl₂10mM HEPES, 10mM glucose, pH 7.0) in Trypan Ultra Blue (ABDBioquest, CA). The quenchers identified above may be determined by quenchers well known in the art such as, for example, bispicramine (DPA), acid Violet 17(AV17), Diazine Black (DB), HLB30818, Trypan blue, bromophenol blue, HLB30701, HLB30702, HLB30703, nitrazine yellow, Nitro Red, DABCYL (molecular probes), QSY (molecular probes), Metal ion quenchers (e.g., Co, molecular probes)²⁺、Ni²⁺、Cu²⁺) And iodide ion displacement. The cell plates and test compounds (0.01. mu.M-100 mM) were placed in a high throughput fluorescence plate reader (Hamamatsu FDSS) that collected images of plate fluorescence before, during, and after the instrument added the test compounds to the cells. Image software (Hamamatsu FDSS) analysis reported changes in relative fluorescence for each well in the cell plate.

Various compounds and extracts (many of which are reported as odorous) were assayed between odor receptor cell lines and in control cells to determine activity and arc-off.

Example 41 consistency of functional response in odorant receptor expressing cell lines

Since the odorant receptor is a G protein-coupled receptor (GPCR), it is important to determine the functional identity of G protein coupling in different cell lines in order to produce meaningful comparisons of responses between different odorant receptors in the presence of different compounds. The relative responses of different cell lines across a range of concentrations of G protein-coupled agonist should be consistent. To validate this, we tested the identity of the G α 15-mediated receptor response of all cell lines expressing different odorant receptors and mouse G α 15 protein in a dose-response curve experiment using different concentrations of isoproterenol, an agonist of 3-adrenergic receptors endogenously expressed in the cells. This endogenous receptor, when stimulated, can couple to expressed G α 15 and induce changes in intracellular free calcium. The percent response was plotted as a function of isoproterenol concentration, and the results were curve-fitted to calculate EC ₅₀Values (concentration of half maximal receptor activation) (see figure 16).

Example 42 identification of broadly modulating, modulating and Selective receptors for functional responsiveness

Cell lines stably expressing the native human odorant receptor gene generated using the method of the invention (i.e., example 39) were tested in functional cell receptor-based studies to detect their activation by a collection of chemically diverse compounds. This allows the identification of broadly, less broadly, and narrowly regulated receptors.

Example 43 identification of receptors activated by a Compound library

Testing the activity of the library of compounds against each of the odorant receptor cell lines. Data regarding receptor activity after addition of compound was generated in a functional cell-based assay and expressed as a percentage activity above the baseline activity of the receptor (i.e., receptor activity when buffer alone was added). The activity of each compound at each receptor was then coded as a highlighted compound with low (< 100% greater than baseline activity; white), medium (101-. The resulting pattern is used to provide a graphical representation of compounds (rows) having selective or broad activity between odorant receptors and receptors (columns) showing broad or selective responses to chemically diverse groups of compounds. Analysis of the data is used to assign values or identify patterns of specific odorant receptors or odorant receptor activities using the test compounds of interest and to identify modulators, including blockers and enhancers. Analysis of this data can also be used to correlate patterns of odorant receptor activity with the physiological effects of the corresponding test odorants.

Example 44 has similar function as an antagonist of agonist-induced odorant receptor Activity Identification of analogs of active compounds

To test the ability to identify analogs of compounds with similar functional activity based on assays that express odorant receptor cells, known modulators or structural analogs of the modulator compounds are tested for their ability to inhibit agonist-induced receptor activity of odorant receptors. The structures of analogs that inhibit the activity of odorant receptors induced by agonists were compared. Thus, cell lines expressing odorant receptors may aid in the discovery of potent odorant receptor antagonists. Modulators with different activities (e.g., potentiation, agonism, blockade, etc.) are also identified by manipulation of the assay and testing and analysis of the results of the assay, which are known to those of ordinary skill in the art.

The embodiments of the invention described above are intended to be merely exemplary and those skilled in the art will understand or be able to ascertain using no more than routine experimentation, many equivalents to the specific procedures described herein. All such equivalent experiments are considered to be within the scope of the present invention and are covered by the following claims. In addition, one of ordinary skill in the art will understand that sequences of operations must be listed in some specific order for explanation and claims, but the invention also includes variations other than such specific orders.

All references cited herein are incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

The claims (modification according to treaty clause 19)

1. A method for isolating a cell endogenously expressing a sweet taste receptor T1R2 subunit and/or a sweet taste receptor T1R3 subunit, wherein the method comprises the steps of:

a. providing a population of cells;

b. introducing into the cell a signaling probe that detects expression of T1R2 and/or introducing into the cell a molecular beacon that detects expression of T1R 3;

c. isolating cells expressing the sweet taste receptor T1R2 subunit and/or the sweet taste receptor T1R3 subunit.

2. A method for isolating cells endogenously expressing a sweet taste receptor T1R2 subunit and a sweet taste receptor T1R3 subunit, wherein the method comprises the steps of:

a. providing a population of cells;

b. introducing a molecular beacon which detects expression of T1R2 into a cell and/or introducing a molecular beacon which detects expression of T1R3 into a cell;

3. The method of claim 1 or 2, wherein the population of known cells does not express T1R2 or T1R 3.

4. The method of claim 1 or 2, wherein any expression level of T1R2 or T1R3 in the isolated cells is at least 10-fold, 50-fold, 100-fold, 250-fold, 500-fold, 750-fold, 1000-fold, 2500-fold, 5000-fold, 7500-fold, 10000-fold, 50000-fold, or at least 100000-fold in the average cell of the population of cells.

5. The method of claim 1 or 2, wherein the genetic variability in the population of cells has been increased prior to the isolating step.

6. An isolated cell produced according to any one of claims 1 to 5.

7. An isolated cell endogenously expressing a sweet taste receptor T1R2 subunit and/or a sweet taste receptor T1R3 subunit, wherein the isolated cell is derived from a population of cells and wherein the expression of the sweet taste receptor T1R2 subunit and/or the sweet taste receptor T1R3 subunit in the isolated cell is at least 10-fold, 50-fold, 100-fold, 250-fold, 500-fold, 750-fold, 1000-fold, 2500-fold, 5000-fold, 7500-fold, 10000-fold, 50000-fold, or at least 100000-fold in the average cell of the population of cells.

8. The cell of claim 7, wherein the population of cells is a population of Chinese Hamster Ovary (CHO) cells, an established neuronal cell line, pheochromocytoma, neuroblastoma fibroblasts, rhabdomyosarcoma cells, dorsal root ganglion cells, NS0 cells, CV-1(ATCC CCL 70), COS-1(ATCC CRL 1650), COS-7(ATCC CRL 1651), CHO-K1(ATCC CCL61), 3T3(ATCC CCL 92), NIH/3T3(ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1(ATCC CCL 26), C-5(ATCC CCL 171), L-cells, HEK-293(ATCC CRL1573), and PCI2(ATCC CRL-1721), HEK293T (ATCC CRL-11268), RBL-SY 8), SH-2265 (cccrl-5Y) (ATCC CRL 1376-1376), ATCC CRL 13735 (ATCC CRL-1721), ATCC CRL 293(ATCC CRL-293, ATCC CRL 1378), MDCK (ATCC CCL-34), SJ-RH30(ATCC CRL-2061), HepG2(ATCC HB-8065), ND7/23(ECACC 92090903), CHO (ECACC85050302), Vero (ATCC CCL 81), Caco-2(ATCC HTB 37), K562(ATCC CCL243), Jurkat (ATCC TIB-152), Per.C6(Crucell, Leiden, The Netherlands), Huvec (ATCC human primary PCS 100-type 010, mouse CRL 2514, CRL 2515, CRL 2516), HuH-7D12 (ECTB 01042712), 293(ATCC CRL 10852), A549(ATCC CCL185), IMR-90(ATCC CCL OS), MCF-7(ATC HTB-22), CHU-2 (CHTB-96), T84(ATCC CCL 248), primary ACC 248 or immortalized cells.

9. A culture of the cell of claim 7.

10. A cell or cell line stably expressing a sweet taste receptor comprising a sweet taste receptor T1R2 subunit and a sweet taste receptor T1R3 subunit, wherein the gene of at least one of the subunits results from the introduction of a nucleic acid encoding the subunit into a host cell or the activation of a gene of a nucleic acid encoding the subunit already present in a host cell, and the cell or cell line is derived from the host cell.

11. A cell or cell line stably expressing a sweet taste receptor comprising a sweet taste receptor T1R2 subunit, a sweet taste receptor T1R3 subunit, and a G protein, wherein expression of at least one of the subunits and the G protein results from introduction of a nucleic acid encoding a subunit or a G protein into a host cell or gene activation of a nucleic acid encoding a subunit or a G protein already present in a host cell, and the cell or cell line is derived from the host cell.

12. The cell or cell line of claim 10 or 11, wherein at least one sweet taste receptor subunit is expressed from a nucleic acid encoding the subunit that is introduced into the host cell.

13. The cell or cell line of claim 10 or 11, wherein at least one sweet taste receptor subunit is expressed from a nucleic acid present in the host cell by gene activation.

14. The cell or cell line of claim 10 or 11, wherein the host cell:

a. is a eukaryotic cell;

b. is a mammalian cell;

c. at least one subunit or G protein that does not endogenously express a sweet taste receptor; or

Any combination of (a), (b), and (c).

15. The cell or cell line of claim 10 or 11, wherein the host cell is a HEK-293 cell.

16. The cell or cell line of claim 10 or 11, wherein the sweet taste receptor

a. Is a mammalian sweet taste receptor;

b. is a human sweet receptor;

c. comprise subunits from different species;

d. comprises one or more subunits that are chimeric;

any combination of (a) - (d).

17. The cell or cell line of claim 10 or 11, wherein the sweet taste receptor is functional.

18. The cell or cell line of claim 10 or 11, which has a Z' value in the assay of at least 0.3.

19. The cell or cell line of claim 17, which has a Z' value in the assay of at least 0.7.

20. The cell or cell line of claim 10 or 11, which stably expresses the sweet taste receptor in the absence of selective pressure in the culture medium.

21. The cell or cell line of claim 10 or 11, wherein the sweet T1R2 receptor subunit is selected from the group consisting of:

a. Comprises the amino acid sequence of SEQ ID NO: 34 or a corresponding amino acid sequence of another species;

b. comprises a nucleotide sequence substantially identical to SEQ ID NO: 34 or a corresponding amino acid sequence of another species having at least 85% identity;

c. comprising a polypeptide consisting of a sequence that hybridizes under stringent conditions to SEQ ID NO: 31 or encoding SEQ ID NO: 34 or a nucleic acid that hybridizes to a nucleic acid of a corresponding amino acid sequence of another species; and

d. comprises a nucleotide sequence consisting of a nucleotide sequence identical to SEQ ID NO: 31 or encoding SEQ ID NO: 34 or a nucleic acid of a corresponding amino acid sequence of another species having at least 85% identity to a nucleic acid encoding the amino acid sequence.

22. The cell or cell line of claim 10 or 11, wherein the sweet taste receptor subunit T1R2 is encoded by a nucleic acid selected from the group consisting of:

a. comprises the amino acid sequence of SEQ ID NO: 31;

b. comprising encoding a polypeptide comprising SEQ ID NO: 34 or a nucleotide sequence of a polypeptide of the corresponding amino acid sequence of another species;

c. a nucleic acid comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid of a) or b); and

d. Comprises a nucleotide sequence substantially identical to SEQ ID NO: 31 or encoding SEQ ID NO: 34 or a nucleic acid of a nucleotide sequence having at least 95% identity to a nucleic acid of the corresponding amino acid sequence of another species.

23. The cell or cell line of claim 10 or 11, wherein the sweet taste receptor T1R3 subunit is selected from the group consisting of:

a. comprises the amino acid sequence of SEQ ID NO: 35 or a corresponding amino acid sequence of another species;

b. comprises a nucleotide sequence substantially identical to SEQ ID NO: 35 or a corresponding amino acid sequence of another species has at least 85% identity to the corresponding amino acid sequence of the other species;

c. comprising a polypeptide consisting of a sequence that hybridizes under stringent conditions to SEQ ID NO: 32 or encoding SEQ ID NO: 35 or a nucleic acid that hybridizes to a nucleic acid of a corresponding amino acid sequence of another species; and

d. comprises a nucleotide sequence consisting of a nucleotide sequence identical to SEQ ID NO: 32 or encoding SEQ ID NO: 35 or a nucleic acid of a corresponding amino acid sequence of another species has at least 85% identity to a nucleic acid encoding the amino acid sequence.

24. The cell or cell line of claim 10 or 11, wherein the sweet taste receptor T1R3 subunit is encoded by a nucleic acid selected from the group consisting of:

a. Comprises the amino acid sequence of SEQ ID NO: 32;

b. comprising encoding a polypeptide comprising SEQ ID NO: 35 or a nucleotide sequence of a polypeptide of the corresponding amino acid sequence of another species;

d. comprises a nucleotide sequence substantially identical to SEQ ID NO: 32 or encoding SEQ ID NO: 35 or a nucleic acid of a nucleotide sequence having at least 85% identity to a nucleic acid of a corresponding amino acid sequence of another species.

25. The cell or cell line of claim 11, wherein the G protein is selected from the group consisting of:

a. comprises the amino acid sequence of SEQ ID NO: 36 or 37 or the corresponding amino acid sequence of another species;

b. comprises a nucleotide sequence substantially identical to SEQ ID NO: 36 or 37 or another species or an amino acid sequence having at least 85% identity to the corresponding amino acid sequence;

c. comprising a polypeptide consisting of a sequence that hybridizes under stringent conditions to SEQ ID NO: 33 or encoding SEQ ID NO: 36 or 37 or a nucleic acid that hybridizes to a nucleic acid of the corresponding amino acid sequence of another species; and

d. comprises a nucleotide sequence consisting of a nucleotide sequence identical to SEQ ID NO: 33 or encoding SEQ ID NO: 36 or 37 or a nucleic acid sequence encoding an amino acid sequence having at least 85% identity to a nucleic acid of the corresponding amino acid sequence of another species.

26. The cell or cell line of claim 11, wherein the G protein is encoded by a nucleic acid selected from the group consisting of:

a. comprises the amino acid sequence of SEQ ID NO: 33;

b. comprises a nucleotide sequence encoding SEQ ID NO: 36 or 37 or a corresponding amino acid sequence of another species;

c. nucleic acids comprising a nucleotide sequence which hybridizes under stringent conditions to the nucleic acid sequence of a) or b);

d. comprises a nucleotide sequence substantially identical to SEQ ID NO: 33 or encoding SEQ ID NO: 36 or 37 or a nucleotide sequence having at least 85% sequence identity to a nucleic acid sequence of the corresponding amino acid sequence of another species.

27. A method for producing a cell or cell line according to claim 10 or 11, comprising the steps of:

a. introducing into a host cell a first vector comprising a nucleic acid encoding a sweet taste receptor T1R2 subunit, a second vector comprising a nucleic acid encoding a sweet taste receptor T1R3 subunit, and optionally a third vector comprising a nucleic acid encoding a G protein;

b. introducing a first molecular beacon detecting expression of the sweet taste receptor T1R2 subunit, a second molecular beacon detecting expression of the sweet taste receptor T1R3 subunit, and optionally a third molecular beacon detecting expression of a G protein into the host cell produced in step a); and

c. Isolating cells expressing the T1R2 subunit, the T1R3 subunit, and optionally the G protein.

28. The method of claim 27, further comprising the step of generating a cell line from the cells isolated in step c).

29. The method of claim 27, wherein the host cell:

a. is a eukaryotic cell;

b. is a mammalian cell;

d.a), b) and c).

30. The method of claim 27, further comprising the steps of:

a. culturing the cells for a period selected from 1 to 4 weeks, 1 to 9 months, or any time therebetween;

b. periodically measuring the expression of the sweet taste receptor or subunit thereof during these time periods, said expression being measured at the RNA or protein level; and

c. selecting a cell or cell line characterized by substantially stable expression of a sweet taste receptor or subunit thereof over a period of time selected from 1 to 4 weeks, 1 to 9 months, or any time therebetween.

31. The method of claim 27, further comprising the steps of:

b. periodically measuring the expression of the sweet taste receptor or subunit thereof during these time periods, measuring the expression at the RNA or protein level; and

c. Selecting cells or cell lines characterized by substantially the same expression level of a sweet taste receptor or subunit thereof over a period of time selected from 1 to 4 weeks, 1 to 9 months, or any time therebetween.

32. The method of claim 31, wherein the measurement of the protein expression level of the sweet taste receptor is performed using a functional assay.

33. The method of claim 27, wherein the T1R2 subunit is selected from the subunits of claims 12a) to d), wherein the T1R3 subunit is selected from the subunits of claims 14a) to d) and wherein the G protein is selected from the proteins of claims 16a) to d).

34. The method of claim 27, wherein the T1R2 subunit encoded by the nucleic acid is selected from the group consisting of the subunits encoded by the nucleic acids of claims 13a) to d), wherein the T1R3 subunit is selected from the group consisting of the subunits encoded by the nucleic acids of claims 15a) to d) and wherein the G protein is selected from the group consisting of the proteins encoded by the nucleic acids of claims 17a) to d).

35. The method of claim 27, wherein the separating step utilizes a fluorescence activated cell sorter.

36. A method for identifying modulators of sweet taste receptor function comprising the steps of contacting at least one cell or cell line of claim 6, 10 or 11 with at least one test compound and detecting a change in sweet taste receptor function.

37. The method of claim 36, wherein the modulator is selected from the group consisting of a sweet taste receptor inhibitor, a sweet taste receptor antagonist, a sweet taste receptor blocker, a sweet taste receptor activator, a sweet taste receptor agonist, or a sweet taste receptor enhancer.

38. The method of claim 36, wherein the sweet taste receptor is a human sweet taste receptor.

39. The method of claim 36, wherein the test compound is a small molecule, chemical moiety, polypeptide, or antibody.

40. The method of claim 36, wherein the test compound is a library of compounds.

41. The method of claim 40, wherein the library is a small molecule library, a combinatorial library, a peptide library, or an antibody library.

42. The method of claim 36, wherein the modulator is selective for enzymatically modified forms of sweet taste receptors.

43. A modulator identified by the method of claim 36.

44. The cell or cell line of claim 6, 10 or 11, wherein the cell or cell line is characterized by substantially identical expression of a sweet taste receptor over a period of time selected from 1 to 4 weeks, 1 to 9 months, or any time therebetween.

45. The cell or cell line of claim 6, 10 or 11, wherein the cell or cell line is characterized by a substantially stable expression level of a sweet taste receptor over a period of time selected from 1 to 4 weeks, 1 to 9 months, or any time therebetween.

46. The cell or cell line of claim 45, wherein said expression level is measured using a functional assay.

47. The cell or cell line of claim 6, 10 or 11, produced by the method of any one of claims 27-35.

48. A method for producing a cell or cell line of claim 10 or 11, wherein said cell has at least one desired property that remains consistent over time, said method comprising the steps of:

a. providing a plurality of cells expressing mRNA encoding a subunit of a sweet taste receptor and optionally a G protein;

b. individually dispersing cells into a single culture vessel, thereby providing a plurality of isolated cell cultures;

c. culturing cells under a desired set of culture conditions using an automated cell culture process characterized by substantially identical conditions for each isolated cell culture, normalizing the number of cells per well in each isolated cell culture during the culturing process, and wherein the isolated cultures are passaged according to the same protocol;

d. determining at least one desired characteristic of the sweet taste receptor or of a cell producing the receptor of the isolated cell culture at least 2 times; and

e. Identifying an isolated cell culture having the desired characteristics in both assays.

49. A cell or cell line that produces a sweet taste receptor and has at least one desired property that remains consistent over time, the cell or cell line produced by the method of claim 48.

50. A cell or cell line stably expressing an umami receptor comprising an umami receptor T1R1 subunit and an umami receptor T1R3 subunit, wherein expression of at least one of the subunits results from introduction of a nucleic acid encoding a subunit into a host cell or gene activation of a nucleic acid encoding a subunit already present in a host cell, and the cell or cell line is derived from the host cell.

51. A cell or cell line stably expressing an umami receptor comprising an umami receptor T1R1 subunit, an umami receptor T1R3 subunit, and a G protein, wherein expression of at least one of the subunit and the G protein results from introduction of a nucleic acid encoding the subunit or the G protein into the host cell or gene activation of a nucleic acid encoding the subunit or the G protein already present in the host cell, and the cell or cell line is derived from the host cell.

52. The cell or cell line of claim 50 or 51, wherein at least one umami receptor subunit is expressed from a nucleic acid encoding said subunit introduced into the host cell.

53. The cell or cell line of claim 50 or 51, wherein at least one umami receptor subunit is expressed from a nucleic acid present in the host cell by gene activation.

54. The cell or cell line of claim 50 or 51, wherein the host cell:

a. is a eukaryotic cell;

b. is a mammalian cell;

c. at least one subunit or G protein that does not endogenously express the umami receptor; or

Any combination of (a), (b), and (c).

55. The cell or cell line of claim 50 or 51, wherein the host cell is a HEK-293 cell.

56. The cell or cell line of claim 50 or 51, wherein the umami receptor

a. Is a mammal;

b. is a human;

c. comprise subunits from different species;

d. comprises one or more subunits that are chimeric;

any combination of (a) - (d).

57. The cell or cell line of claim 50 or 51, wherein said umami receptor is functional.

58. The cell or cell line of claim 50 or 51 having a Z' value in the assay of at least 0.3.

59. The cell or cell line of claim 58, which has a Z' value in the assay of at least 0.7.

60. The cell or cell line of claim 50 or 51, which stably expresses the umami receptor in culture in the absence of selective pressure.

61. The cell or cell line of claim 50 or 51, wherein the umami T1R1 receptor subunit is selected from the group consisting of:

a. comprises a sequence selected from SEQ ID NO: 42-45 and a corresponding amino acid sequence of another species;

b. comprising a nucleotide sequence substantially identical to a sequence selected from SEQ ID NO: 42-45 and a corresponding amino acid sequence of another species having at least 85% identity;

c. comprising a polypeptide consisting of a sequence that hybridizes under stringent conditions to SEQ ID NO: 41 or encodes a polypeptide selected from the group consisting of SEQ ID NO: 42-45 or a nucleic acid that hybridizes to a nucleic acid of a corresponding amino acid sequence of another species; and

d. comprises a nucleotide sequence consisting of a nucleotide sequence identical to SEQ ID NO: 41 or encoding SEQ ID NO: 42-45 or a nucleic acid of a corresponding amino acid sequence of another species, has at least 85% identity to a nucleic acid encoding the amino acid sequence.

62. The cell or cell line of claim 50 or 51, wherein the umami receptor subunit T1R1 is encoded by a nucleic acid selected from the group consisting of:

a. comprises the amino acid sequence of SEQ ID NO: 41;

b. comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 42-45 or a nucleotide sequence of a polypeptide of the corresponding amino acid sequence of another species;

d. comprises a nucleotide sequence substantially identical to SEQ ID NO: 41 or encodes a polypeptide selected from SEQ ID NO: 42-45 or a nucleic acid of a nucleotide sequence that is at least 95% identical to a nucleic acid of a corresponding amino acid sequence of another species.

63. The cell or cell line of claim 50 or 51, wherein the umami receptor T1R3 subunit is selected from the group consisting of:

b. comprises a nucleotide sequence substantially identical to SEQ ID NO: 35 or a corresponding amino acid sequence of another species has at least 85% identity to the umami receptor subunit;

64. The cell or cell line of claim 50 or 51, wherein the sweet taste receptor T1R3 subunit is encoded by a nucleic acid selected from the group consisting of:

a. comprises the amino acid sequence of SEQ ID NO: 32;

65. The cell or cell line of claim 51, wherein said G protein is selected from the group consisting of:

66. The cell or cell line of claim 51, wherein said G protein is encoded by a nucleic acid selected from the group consisting of:

a. comprises the amino acid sequence of SEQ ID NO: 33 or another species;

67. A method for producing the cell or cell line of claim 50 or 51, comprising the steps of:

a. introducing into a host cell a first vector comprising a nucleic acid encoding an umami receptor T1R1 subunit, a second vector comprising a nucleic acid encoding an umami receptor T1R3 subunit, and optionally a third vector comprising a nucleic acid encoding a G protein;

b. Introducing a first molecular beacon detecting expression of the umami receptor T1R1 subunit, a second molecular beacon detecting expression of the umami receptor T1R3 subunit and optionally a third molecular beacon detecting expression of a G protein into the host cell produced in step a); and

c. isolating cells expressing the T1R1 subunit, the T1R3 subunit, and optionally the G protein.

68. The method of claim 67, further comprising the step of generating a cell line from the cells isolated in step c).

69. The method of claim 67, wherein said host cell:

a. is a eukaryotic cell;

b. is a mammalian cell;

d.a), b) and c).

70. The method of claim 67, further comprising the steps of:

b. periodically measuring the expression of said umami receptor or subunit thereof during these time periods, said expression being measured at the RNA or protein level; and

c. selecting cells or cell lines characterized by substantially the same expression of the umami receptor or subunit thereof over a period of time selected from 1 to 4 weeks, 1 to 9 months, or any time therebetween.

71. The method of claim 67, further comprising the steps of:

b. periodically measuring the expression of the umami receptor or subunit thereof during these time periods, measuring the expression at the RNA or protein level; and

c. selecting cells or cell lines characterized by substantially the same expression level of the umami receptor or subunit thereof over a period of time selected from 1 to 4 weeks, 1 to 9 months, or any time therebetween.

72. The method of claim 71, wherein the measuring of the protein expression level of the umami receptor is performed using a functional assay.

73. The method of claim 67, wherein the T1R1 subunit is selected from the subunits of claims 12a) to d), wherein the T1R3 subunit is selected from the subunits of claims 14a) to d) and wherein the G protein is selected from the proteins of claims 16a) to d).

74. The method of claim 67, wherein the T1R1 subunit encoded by the nucleic acid is selected from the group consisting of the subunits encoded by the nucleic acids of claims 13a) to d), wherein the T1R3 subunit is selected from the group consisting of the subunits encoded by the nucleic acids of claims 15a) to d) and wherein the G protein is selected from the group consisting of the proteins encoded by the nucleic acids of claims 17a) to d).

75. The method of claim 67, wherein said separating step utilizes a fluorescence activated cell sorter.

76. A method for identifying a modulator of umami receptor function comprising the steps of contacting at least one cell or cell line of claim 50 or 51 with at least one test compound and detecting a change in the umami receptor function.

77. The method of claim 76, wherein said modulator is selected from the group consisting of an umami receptor inhibitor, an umami receptor antagonist, an umami receptor blocker, an umami receptor activator, an umami receptor agonist, and an umami receptor potentiator.

78. The method of claim 76, wherein the umami receptor is a human umami receptor.

79. The method of claim 76, wherein the test compound is a small molecule, chemical moiety, polypeptide, or antibody.

80. The method of claim 76, wherein the test compound is a library of compounds.

81. The method of claim 80, wherein the library is a small molecule library, a combinatorial library, a peptide library, or an antibody library.

82. The method of claim 76, wherein the modulator is selective for an enzymatically modified form of the umami receptor.

83. A modulator identified by the molecule of claim 76.

84. The cell or cell line of claim 50 or 51, wherein said cell or cell line is characterized by substantially stable expression of the umami receptor over a period of time selected from 1 to 4 weeks, 1 to 9 months, or any time therebetween.

85. The cell or cell line of claim 50 or 51, wherein said cell or cell line is characterized by substantially the same expression of the umami receptor over a period of time selected from 1 to 4 weeks, 1 to 9 months, or any time therebetween.

86. The cell or cell line of claim 85, wherein said expression level is measured using a functional assay.

87. The cell or cell line of claim 50 or 51, produced by the method of any one of claims 18-26.

88. A method for producing the cell or cell line of claim 50 or 51, wherein said cell has at least one desired property that remains consistent over time, said method comprising the steps of:

a. providing a plurality of cells expressing mRNA encoding the subunits of the umami receptor and optionally the G protein;

d. Determining at least one desired characteristic of the umami receptor or of a cell producing the receptor of the isolated cell culture at least 2 times; and

89. A cell or cell line that produces an umami receptor and has at least one desired property that remains consistent over time, the cell or cell line produced by the method of claim 88.

90. A cell or cell line engineered to stably express a bitter taste receptor.

91. The cell or cell line of claim 90, wherein the bitter taste receptor is expressed from a nucleic acid introduced into the cell or cell line.

92. The cell or cell line of claim 90, wherein the bitter taste receptor is expressed from an endogenous nucleic acid by engineered gene activation.

93. The cell or cell line of claim 90, wherein the cell or cell line stably expresses at least one other bitter taste receptor.

94. The cell or cell line of claim 93, wherein at least one other bitter receptor is endogenously expressed.

95. The cell or cell line of claim 93, wherein at least one other bitter taste receptor is expressed from a nucleic acid introduced into the cell or cell line.

96. The cell or cell line of claim 95, wherein the bitter taste receptor and at least one other bitter taste receptor are expressed from an isolated nucleic acid introduced into the cell or cell line.

97. The cell or cell line of claim 95, wherein the bitter taste receptor and the at least one other bitter taste receptor are both expressed from a single nucleic acid introduced into the cell or cell line.

98. The cell or cell line of claim 90, wherein the cell or cell line stably expresses:

a. an endogenous G protein;

b. a heterologous G protein or

c. And both.

99. The cell or cell line of claim 98, wherein the G protein is a heteromultimeric G protein comprising 3 different subunits, and wherein at least 2 different subunits are expressed from different nucleic acids introduced into the cell or cell line.

100. The cell or cell line of claim 98, wherein the G protein is a heteromultimeric G protein comprising 3 different subunits, and wherein all 3 different subunits are expressed from the same nucleic acid introduced into the cell or cell line.

101. The cell or cell line of claim 90, wherein the cell or cells in the cell line are:

a. a eukaryotic cell;

b. a mammalian cell;

c. Cells that do not express endogenous bitter taste receptors; or

Any combination of (a), (b), and (c).

102. The cell or cell line of claim 90, wherein said cell or cell in said cell line is a human embryonic kidney 293T cell.

103. The cell or cell line of claim 90, wherein the bitter taste receptor:

a. is a mammal;

b. is a human;

c. (ii) does not have a polypeptide tag at its amino terminus or carboxy terminus;

any combination of (a), (b), and (c).

104. The cell or cell line of claim 90, wherein said cell or cell line produces a Z' value of at least 0.45 in an assay selected from the group consisting of: cell-based assays, fluorescent cell-based assays, high throughput screening assays, reporter cell-based assays, G protein-mediated cell-based assays, and calcium flux cell-based assays.

105. The cell or cell line of claim 90, wherein said cell or cell line produces a Z' value of at least 0.5 in an assay selected from the group consisting of: cell-based assays, fluorescent cell-based assays, high throughput screening assays, reporter cell-based assays, G protein-mediated cell-based assays, and calcium flux cell-based assays.

106. The cell or cell line of claim 90, wherein said cell or cell line produces a Z' value of at least 0.6 in an assay selected from the group consisting of: cell-based assays, fluorescent cell-based assays, high throughput screening assays, reporter cell-based assays, G protein-mediated cell-based assays, and calcium flux cell-based assays.

107. The cell or cell line of claim 90, wherein the cell or cell line stably expresses the bitter taste receptor in antibiotic-free medium for a period of time selected from the group consisting of at least 2 weeks, at least 4 weeks, at least 6 weeks, at least 3 months, at least 6 months, and at least 9 months.

108. The cell or cell line of claim 90, wherein the bitter taste receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NO:

seq ID NOS: any one of 77-101;

b. and SEQ ID NOS: 77-101, having at least 95% identity to the amino acid sequence of any one of seq id no;

c. comprising a polypeptide synthesized by reacting a polypeptide comprising SEQ ID NOS: 51-75, or a nucleic acid sequence encoding an amino acid sequence that hybridizes to a nucleic acid of the reverse complement of any one of 51-75; and

d. the polypeptide encoded by a nucleic acid sequence set forth as SEQ ID NOS: 51-75, or a variant allele thereof.

109. The cell or cell line of claim 90, wherein the bitter taste receptor comprises an amino acid sequence encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:

seq ID NOS: any one of 51-75;

b. and SEQ ID NOS: 51-75, or a nucleotide sequence at least 95% identical to any one of the above; and

c. under stringent conditions with a nucleic acid sequence comprising SEQ ID NOS: 51-75, or a sequence of a nucleic acid that hybridizes to a nucleic acid of the reverse complement of any one of 51-75; and

d. As SEQ ID NOS: 51-75, or a variant thereof.

110. The cell or cell line of claim 90, wherein the bitter taste receptor is a functional bitter taste receptor.

111. The cell or cell line of claim 90, wherein the cell or cell line changes in intracellular free calcium concentration when contacted with isoproterenol.

112. The cell or cell line of claim 111, wherein the isoproterenol has an EC50 value of about 1nM to about 20nM in a dose response curve performed with the cell or cell line.

113. The cell or cell line of claim 90, wherein said cell or cell line has a signal to noise ratio of greater than 1.

114. The collection of cells or cell lines of claim 90, wherein the collection comprises 2 or more cells or cell lines, each cell or cell line stably expressing a different bitter taste receptor or allelic variant thereof.

115. The collection of claim 114, wherein the collection additionally comprises cells or cell lines engineered to stably express bitter taste receptors with known ligands.

116. The collection of claim 114 wherein the allelic variants thereof are SNPs.

117. The collection of claim 114, wherein each of the cells or cell lines changes in intracellular free calcium concentration when contacted with isoproterenol.

118. The collection of claim 117, wherein the isoproterenol has an EC50 value of about 1nM to about 20nM in a dose-response curve performed with each cell or cell line.

119. The collection of claim 114, wherein the cells or cell lines are matched to share the same physiological properties to allow parallel processing.

120. The collection of claim 119, wherein the physiological property is growth rate.

121. The collection of claim 119, wherein the physiological property is adhesion to a tissue culture surface.

122. The collection of claim 119 wherein the physiological property is a Z' factor.

123. The collection of claim 119, wherein the physiological property is the expression level of a bitter taste receptor.

124. The collection of cells or cell lines of claim 90, wherein the collection comprises 2 or more cells or cell lines, each cell or cell line stably expressing the same bitter taste receptor or allelic variant thereof.

125. The collection of claim 124, wherein the collection additionally comprises cells or cell lines engineered to stably express bitter taste receptors with known ligands.

126. The collection of claim 124 wherein the allelic variants thereof are SNPs.

127. The collection of claim 124, wherein each of the cells or cell lines changes in intracellular free calcium concentration when contacted with isoproterenol.

128. The collection of claim 127, wherein the isoproterenol has an EC50 value of about 1nM to about 20nM in a dose-response curve performed with each cell or cell line.

129. The collection of claim 124, wherein the cells or cell lines are matched to share the same physiological properties, thereby allowing parallel processing.

130. The collection of claim 129, wherein the physiological property is growth rate.

131. The collection of claim 199, wherein the physiological property is adhesion to a tissue culture surface.

132. The collection of claim 129, wherein the physiological property is a Z' factor.

133. The collection of claim 129, wherein the physiological property is the expression level of a bitter taste receptor.

134. A method of producing a cell stably expressing a bitter taste receptor comprising:

a. introducing a nucleic acid encoding a bitter taste receptor into a plurality of cells;

b. introducing a molecular beacon that detects expression of a bitter taste receptor into the plurality of cells provided in step (a); and

c. isolating cells expressing the bitter taste receptor.

135. The method of claim 134, further comprising the step of generating a cell line from the cells isolated in step (c).

136. The method of claim 135, wherein the cell line produced stably expresses the bitter taste receptor in antibiotic-free medium for a period of time selected from the group consisting of at least 2 weeks, at least 4 weeks, at least 6 weeks, at least 3 months, at least 6 months, and at least 9 months.

137. The method of claim 134, wherein the cell is:

a. a eukaryotic cell;

b. a mammalian cell;

c. cells that do not express endogenous bitter taste receptors; or

Any combination of (a), (b), and (c).

138. The method of claim 134, wherein the cells in the cell line are human embryonic kidney 293T cells.

139. The method of claim 134, wherein the bitter taste receptor:

a. is a mammal;

b. is a human;

any combination of (a), (b), and (c).

140. The method of claim 134, wherein the bitter taste receptor comprises an amino acid sequence selected from the group consisting of seq id nos:

seq ID NOS: any one of 77-101;

141. The method of claim 134, wherein the bitter taste receptor comprises an amino acid sequence encoded by a nucleotide sequence selected from the group consisting of seq id nos:

Seq ID NOS: any one of 51-75;

d. as SEQ ID NOS: 51-75, or a variant thereof.

142. The method of claim 134, wherein the bitter taste receptor is a functional bitter taste receptor.

143. The method of claim 134, wherein the cells isolated in step (c) have a change in intracellular free calcium concentration when contacted with isoproterenol.

144. The method of claim 143, wherein the isoproterenol has an EC50 value of about 1nM to about 20nM in a dose response curve performed with the cells.

145. The method of claim 143, wherein said separating utilizes a fluorescence activated cell sorter.

146. The method of claim 143, wherein the cell stably expresses an endogenous G protein, a heterologous G protein, or both.

147. The method of claim 143, further comprising introducing into the cell a nucleic acid encoding a G protein:

a. prior to introducing the nucleic acid encoding the bitter taste receptor;

b. After introducing the nucleic acid encoding the bitter taste receptor; or

c. Simultaneously with the introduction of a nucleic acid encoding said bitter taste receptor.

148. The method of claim 143, wherein the nucleic acid encoding the bitter taste receptor and the nucleic acid encoding the G protein are on a single vector.

149. The method of claim 58, further comprising introducing into the cell a molecular beacon that detects expression of the G protein:

a. prior to introducing a molecular beacon that detects expression of the bitter taste receptor;

b. after introducing a molecular beacon that detects expression of the bitter taste receptor; or

c. Simultaneously with the introduction of a molecular beacon that detects the expression of the bitter taste receptor.

150. The method of claim 149, wherein the molecular beacon that detects expression of the bitter taste receptor and the molecular beacon that detects expression of the G protein are different molecular beacons.

151. The method of claim 58, further comprising:

a. prior to isolating cells expressing the bitter taste receptor;

b. after isolating cells expressing the bitter taste receptor; or

c. Simultaneously with isolating the cells expressing the bitter taste receptor;

isolating cells expressing the G protein, thereby isolating cells expressing the bitter taste receptor and the G protein.

152. A method of identifying a modulator of bitter receptor function, comprising:

a. Contacting a cell or cell line stably expressing a bitter taste receptor with a test compound; and

b. detecting a change in function of the bitter taste receptor.

153. The method of claim 152, wherein said detecting utilizes an assay that measures intracellular free calcium.

154. The method of claim 153, wherein intracellular free calcium is measured using one or more calcium-sensitive fluorescent dyes, a fluorescence microscope, and optionally a fluorescent plate reader, and wherein at least one fluorescent dye binds free calcium.

155. The method of claim 153, wherein the intracellular free calcium is monitored by real-time imaging using one or more calcium-sensitive fluorescent dyes, and wherein at least one fluorescent dye binds free calcium.

156. The method of claim 152, wherein said cell or a cell in said cell line is:

a. a eukaryotic cell;

b. a mammalian cell;

c. cells that do not express any endogenous bitter taste receptors; or

Any combination of (a), (b), and (c).

157. The method of claim 152, wherein the cells in the cell line are human embryonic kidney 293T cells.

158. The method of claim 152, wherein the bitter taste receptor:

a. is a mammal;

b. is a human;

c. (ii) does not have a polypeptide tag at its amino terminus or carboxy terminus; or

Any combination of (a), (b), and (c).

159. The method of claim 152, wherein the bitter taste receptor comprises an amino acid sequence selected from the group consisting of seq id nos:

seq ID NOS: any one of 77-101;

160. The method of claim 152, wherein the bitter taste receptor comprises an amino acid sequence encoded by a nucleotide sequence selected from the group consisting of seq id nos:

seq ID NOS: any one of 51-75;

d. as SEQ ID NOS: 51-75 of an allelic variant of any of the nucleic acid sequences.

161. The method of claim 152, wherein the cell or cell line changes in intracellular free calcium concentration when contacted with isoproterenol.

162. The method of claim 161, wherein the isoproterenol has an EC50 value of about 1nM to about 20nM in a dose response curve performed with the cell or cell line.

163. The method of claim 152, wherein the test compound is a bitter taste receptor inhibitor.

164. The method of claim 153, further comprising contacting said cell or cell line with a known agonist of bitter said taste receptor prior to or simultaneously with the step of contacting said cell or cell line with said test compound.

165. The method of claim 152, wherein the test compound is a bitter receptor agonist.

166. The method of claim 165, further comprising contacting the cell or cell line with a known inhibitor of the bitter taste receptor prior to or simultaneously with the step of contacting the cell or cell line with the test compound.

167. The method of claim 152, wherein the test compound is a small molecule, chemical moiety, polypeptide, antibody or food extract.

168. A method of identifying a modulator of bitter receptor function, comprising:

a. contacting a collection of cell lines with a library of different test compounds, wherein the collection of cell lines comprises 2 or more cell lines, each cell line stably expressing the same bitter taste receptor or allelic variant thereof, and wherein each cell line is contacted with one or more test compounds in the library; and

b. Detecting a change in function of the bitter taste receptor or allelic variant thereof stably expressed by each cell line.

169. The method of claim 168, wherein said detecting utilizes an assay that measures or monitors free calcium within a cell.

170. The method of claim 169, wherein intracellular free calcium is measured using one or more calcium-sensitive fluorescent dyes, a fluorescence microscope, and optionally a fluorescent plate reader, and wherein at least one fluorescent dye binds free calcium.

171. The method of claim 169, wherein the intracellular free calcium is monitored by real-time imaging using one or more calcium-sensitive fluorescent dyes, and wherein at least one fluorescent dye binds free calcium.

172. The method of claim 168, wherein the library is a small molecule library, a combinatorial library, a peptide library, or an antibody library.

173. The method of claim 168 wherein the test compounds are small molecules, chemical moieties, polypeptides, antibodies and food extracts.

174. The method of claim 168, further comprising contacting the collection of cell lines with a known bitter taste receptor agonist or inhibitor prior to or simultaneously with step (a).

175. A method of identifying a modulator of bitter receptor function, comprising:

a. Contacting a collection of cell lines with a test compound, wherein the collection of cell lines comprises 2 or more cell lines, each cell line stably expressing a different bitter taste receptor or allelic variant thereof; and

b. changes in the function of the bitter taste receptor stably expressed by each cell line were examined.

176. The method of claim 175, wherein the detecting utilizes an assay that measures or monitors free calcium within the cell.

177. The method of claim 176, wherein the intracellular free calcium is measured using one or more calcium-sensitive fluorescent dyes, a fluorescence microscope, and optionally a fluorescent plate reader, and wherein at least one fluorescent dye binds free calcium.

178. The method of claim 176, wherein the intracellular free calcium is monitored by real-time imaging using one or more calcium-sensitive fluorescent dyes, wherein at least one fluorescent dye binds to free calcium.

179. The method of claim 175, wherein the test compound is selected from the group consisting of a small molecule, a chemical moiety, a polypeptide, an antibody, and a food extract.

180. The method of claim 175, further comprising contacting the collection of cell lines with a known bitter taste receptor agonist or inhibitor prior to or simultaneously with step (a).

181. A cell engineered to stably express a bitter taste receptor at a consistent level over time, the cell prepared by a method comprising:

a. providing a plurality of cells expressing mRNA encoding the bitter taste receptor;

c. culturing the cells under a desired set of culture conditions using an automated cell culture method, the culture method characterized by substantially identical conditions for each isolated cell culture, normalizing the number of cells in each isolated cell culture during the culturing, and wherein the isolated cultures are passaged according to the same protocol;

d. assaying the isolated cell culture to measure expression of the bitter taste receptor at least 2 times; and

e. identifying an isolated cell culture that expresses the bitter taste receptor at a consistent level in both assays, thereby obtaining the cell.

182. Matching a combination of clonal cell lines to a subject group, wherein the clonal cell lines are of the same type, and wherein at least 2 cell lines in the subject group express different combinations of subunits of a multi-subunit protein of interest; and wherein the clonal cell lines of the subject panel are matched such that they are cultured in parallel under the same cell culture conditions.

183. The matched panel of clonal cell lines of claim 182, wherein the cell line is selected from the group consisting of primary cells and immortalized cells.

184. The matched panel of clonal cell lines of claim 182, wherein the clonal cell line cells are eukaryotic cells and are selected from the group consisting of: fungal cells, insect cells, mammalian cells, yeast cells, algae, crustacean cells, arthropod cells, avian cells, reptilian cells, amphibian cells, plant cells, humans, non-human primates, bovines, porcines, felines, rats, marsupials, murines, canines, ovines, caprines, rabbits, guinea pigs, hamsters.

185. The matched panel of clonal cell lines of claim 182, wherein the cells in the cell line are engineered to express the protein of interest.

186. The matched panel of clonal cell lines of claim 182, wherein cells in the cell line express the protein of interest from an introduced nucleic acid that encodes the protein or, in the case of a multimeric protein, a protein subunit.

187. The matched panel of clonal cell lines of claim 182, wherein the cells express the protein of interest from an endogenous nucleic acid and wherein the cells are engineered to activate transcription of an endogenous protein, or in the case of a multimeric protein, of a protein subunit.

188. The matched panel of clonal cell lines of claim 182, wherein the panel comprises at least 4, at least 6, or at least 20 clonal cell lines.

189. The matched panel of clonal cell lines of claim 182, wherein the multi-subunit protein is selected from the group consisting of: ion channels, G protein-coupled receptors (GPCRs), tyrosine receptor kinases, cytokine receptors, nuclear steroid hormone receptors, antibodies, biologicals, and immunoreceptors.

190. A cell that expresses at least one RNA of interest, wherein said RNA of interest is encoded by an introduced nucleic acid, said cell being characterized in that it produces the RNA of interest in a form that is or is capable of becoming biologically active, wherein said cell is cultured in the absence of selective pressure and wherein the expression of RNA does not change more than 30% within 3 months.

191. The cell of claim 190, wherein the expression of said RNA does not change by more than 30% within 6 months.

192. A cell expressing at least one protein of interest, wherein the protein of interest is encoded by an introduced nucleic acid, said cell being characterized in that it produces the protein of interest in a form that is or is capable of becoming biologically active, wherein said cell is cultured in the absence of selective pressure and wherein the expression of said protein does not change by more than 30% within 3 months.

193. The cell of claim 192, wherein the expression of said protein does not change by more than 30% within 6 months.

194. A cell expressing at least one protein of interest, wherein the protein of interest does not have a known ligand or wherein there is no known assay to detect functional expression of the protein of interest; and wherein the protein of interest does not comprise a protein tag.

195. A cell that expresses at least one protein of interest from an introduced nucleic acid encoding the at least one protein of interest, wherein the at least one protein of interest alters a physiological characteristic of the cell, and wherein the physiological characteristic of the cell does not change by more than 25% within 3 months under constant cell culture conditions.

196. A cell that expresses a protein of interest, wherein the cell is engineered to activate transcription of an endogenous nucleic acid encoding the protein of interest, wherein the protein of interest alters a physiological characteristic of the cell, and wherein the physiological characteristic of the cell does not change by more than 25% over 3 months under constant cell culture conditions.

197. A cell that expresses an RNA of interest, wherein the RNA of interest is encoded by the introduced nucleic acid, wherein at least one RNA of interest alters a physiological property of the cell, and wherein the physiological property of the cell does not change by more than 25% within 3 months under constant cell culture conditions.

198. A cell that expresses at least one protein of interest from an introduced nucleic acid encoding the at least one protein of interest, said cell being characterized in that it produces the protein of interest in a form that is or is capable of becoming biologically active, and wherein said cell consistently and reproducibly expresses at least 500, 2,500, 5,000, or 100,000 picograms of protein per cell per day.

199. A cell that expresses a protein of interest, wherein the cell is engineered to activate transcription of an endogenous nucleic acid encoding the protein of interest, the cell being characterized in that it produces the protein of interest in a form that is or is capable of becoming biologically active, and wherein the cell consistently and reproducibly expresses at least 500, 2,500, 5,000, or 100,000 picograms of protein per cell per day.

200. The cell of any one of claims 195-199, wherein the cell is produced in a time period selected from less than 1 week, less than 2 weeks, less than 3 weeks, less than 4 weeks, less than 1 month, less than 2 months, less than 3 months, less than 4 months, less than 5 months, less than 6 months, less than 7 months, less than 8 months, or less than 9 months.

201. A cell that expresses at least one protein of interest from an introduced nucleic acid encoding the at least one protein of interest, said cell being characterized in that it produces the protein of interest in a biologically active or capable of becoming biologically active form, wherein said cell is produced in a time period selected from the group consisting of less than 7 months, less than 8 months or less than 9 months, and wherein said cell consistently and reproducibly expresses at least 0.5, 1.0, 5.0 or 10g/L of protein.

202. A cell that expresses a protein of interest, wherein the cell is engineered to activate transcription of an endogenous nucleic acid encoding the protein of interest, the cell being characterized in that it produces the protein of interest in a form that is or is capable of becoming biologically active, wherein the cell is produced in a time period selected from less than 7 months, less than 8 months, or less than 9 months, and wherein the cell consistently and reproducibly expresses at least 0.5, 1.0, 5.0, or 10g/L of the protein.

203. The cell of claim 201 or 202, wherein the cell is produced in a time period selected from less than 3 months, less than 4 months, or less than 6 months.

204. The cell of any one of claims 195-203, wherein the protein is a monomeric protein.

205. The cell of any one of claims 195-203, wherein the protein is a multimeric protein.

206. The cell of any one of claims 195-203, wherein the protein of interest does not comprise a protein tag or the cell is cultured in the absence of selective pressure, or a combination thereof.

207. The cell of claim 205, wherein said multimeric protein of interest comprises at least 2, 3, 4, 5, or at least 6 subunits.

208. The cell of claim 205, wherein said multimeric protein of interest is selected from the group consisting of an ion channel, a G protein-coupled receptor (GPCR), a tyrosine receptor kinase, a cytokine receptor, a nuclear steroid hormone receptor, an antibody, a biologic, and an immunological receptor.

209. The cell of claim 205, wherein the multimeric protein of interest is an ion channel and the cell physiological property is selected from the group consisting of membrane potential, UPR, cell viability, ability to increase protein yield, folding assembly, secretion, integration into the cell membrane, post-translational modification, glycosylation, or any combination thereof.

210. A cell line produced from the cell of any one of claims 195-209.

211. A method of identifying a modulator of a protein of interest, comprising the steps of:

a. contacting the cell of any one of claims 195 to 210 with a test compound; and

b. detecting a change in activity of the protein of interest in cells contacted with the test compound as compared to the activity of the protein in cells not contacted with the test compound; wherein a compound that produces a difference in activity in the presence as compared to the absence is a modulator of the protein of interest.

212. A matched panel of cells or clonal cell lines comprising at least 2 cells according to claim 195-209 or 2 clonal cell lines of claim 210, wherein said at least 2 cells or said at least 2 clonal cell lines are matched such that they are cultured in parallel under the same cell culture conditions.

213. The matched panel of claim 212, wherein the matched panel comprises at least 10 cells of claim 195-209 or 10 clonal cell lines of claim 210, and the at least 10 cells or 10 clonal cell lines are matched such that they are cultured in parallel under the same cell culture conditions.

214. The matched panel of claim 213, wherein the panel comprises at least 100 cells of claim 195-209 or at least 100 of claim 210 and the at least 100 cells or the at least 100 clonal cell lines are cultured in parallel under the same cell culture conditions.

215. A matched panel of clonal cell lines, wherein the clonal cell lines are of the same type and comprise a first and a second protein of interest; wherein the first protein of interest is the same in each clonal cell line; wherein the second protein of interest is a component of a functional biological pathway; and wherein:

a. Said group of subjects comprises at least 5 cell lines;

b. generating the subject group in less than 6 months;

c. the first and second proteins of interest do not have a protein tag;

d. culturing the clonal cell line in the absence of selective pressure; or

Any combination of e.a) -d).

216. The matched panel of claim 215, wherein the first protein of interest is an antibody and the functional biological pathway is a glycosylation pathway.

217. A method for generating an in vitro correlation of a physiological property in vivo, wherein the method comprises:

a. contacting a compound or compounds having a physiological property with a first cell expressing a first protein of interest;

b. determining the effect of the compound or compounds on the first protein in a functional assay;

c. contacting the compound or compounds with a second cell expressing a second protein of interest;

d. determining the effect of the compound or compounds on the second protein in a functional assay;

wherein the first and second proteins independently i) do not comprise a protein tag; ii) consistently and reproducibly produced in a form suitable for use in a functional assay such that the cells have a Z' factor of at least 0.4 in the functional assay, iii) are expressed in cells cultured in the absence of selective pressure, iv) alter the physiological properties of the cells and wherein the physiological properties of the cells do not change by more than 25% within 3 months under constant cell culture conditions; v) is stably expressed in cells cultured in the absence of selection and wherein the expression of the protein does not change by more than 30% within 3 months, vi) is expressed in cells that also express another protein and are cultured in the absence of selection pressure or vii) any combination thereof; and

Wherein the profiles obtained in steps a) to d) provide an in vitro correlation of physiological properties in vivo.

218. The method of claim 217, wherein the first and second proteins of interest are independently selected from monomeric or multimeric proteins.

219. The method of claim 218, wherein the multimeric protein comprises at least 2, 3, 4, 5, or 6 subunits.

220. The method of claim 219, wherein the multimeric protein is a heteromultimeric protein.

221. The method of any one of claims 217-219, wherein:

a. the first and second cells are within a subject group of cells that also includes at least one other cell;

b. each cell in the subject set of cells is engineered to express a different protein and the cell is contacted with a compound or compounds;

c. determining the effect of the compound or compounds on each protein expressed in each cell in the subject set of cells in a functional assay; and

d. the activity profile of the compound or compounds in each cell is used to generate in vitro correlations of physiological properties.

222. The method of claim 221, wherein each protein is independently selected from a monomeric protein or a multimeric protein.

223. The method of claim 222, wherein said multimeric protein comprises at least 2, 3, 4, 5, or 6 subunits.

224. The method of claim 223, wherein the multimeric protein is a heteromultimeric protein.

225. A method for predicting a physiological property of a test compound, wherein the method comprises:

a. contacting the test compound or compounds with a first cell expressing a first protein of interest according to claim 217-220;

b. determining the effect of the test compound or compounds on the first protein in a functional assay;

c. contacting a test compound or a plurality of said test compounds with a second cell expressing a second protein of interest according to claim 217-220;

d. determining the effect of the test compound or compounds on the second protein in a functional assay;

e. comparing the activity profile of the test compound obtained in steps a) to d) with the in vitro correlation as generated by the method of claim 217.

Wherein the first and second proteins independently i) do not comprise a protein tag, ii) are consistently and reproducibly produced in a form suitable for use in a functional assay such that the cell has a Z' factor of at least 0.4 in the functional assay, iii) are expressed in cells cultured in the absence of selective pressure, iv) alter the physiological properties of the cell and wherein the physiological properties of the cell do not change by more than 25% within 3 months under constant cell culture conditions; v) is stably expressed in cells cultured in the absence of selective pressure and wherein the expression of the protein does not change by more than 30% within 3 months, vi) is expressed in cells that also express another protein and are cultured in the absence of selective pressure or vii) any combination thereof; and

Wherein the test compound or compounds is/are predicted to have an in vitro relevant physiological property if the activity profile of the test compound is at least 90% identical to the in vitro relevant activity profile.

226. A method for confirming a physiological property of a test compound or test compounds, wherein the method comprises:

a. contacting the test compound or compounds with a first cell expressing a first protein of interest according to claim 217;

c. contacting the test compound or compounds with a second cell expressing a second protein of interest according to claim 217;

e. comparing the activity profile of the test compound or compounds obtained in steps a) to d) with the in vitro correlation of physiological properties as generated by the method of claim 217,

Wherein a test compound or compounds is/are confirmed to have a physiological property if its activity profile is at least 90% identical to that of an in vitro correlation.

227. The method of any one of claims 225-226, wherein the first and second proteins are independently selected from the group consisting of monomeric proteins and multimeric proteins.

228. The method of claim 227, wherein said multimeric protein comprises at least 2, 3, 4, 5, or at least 6 subunits.

229. The method of claim 228, wherein said multimeric protein is a heteromultimeric protein.

230. The method as set forth in any one of claims 225-229 wherein:

a. the first and second cells are cells in a subject group of cells that further includes at least one other cell;

b. each cell in the subject group of cells is engineered to express a different protein and the cell is contacted with the test compound or compounds;

c. determining the effect of the test compound or compounds on each protein of interest expressed in each cell of the subject set of cells in a functional assay; and

d. The activity profile of the test compound or compounds in each cell is used to compare with the profile of in vitro correlations.

231. The method of claim 230, wherein each protein is independently selected from a monomeric protein or a multimeric protein.

232. The method of claim 231, wherein said multimeric protein comprises at least 2, 3, 4, 5, or at least 6 subunits.

233. The method of claim 233, wherein the multimeric protein is a heteromultimeric protein.

234. The method of any one of claims 217, 221, 225, 226 or 230, wherein at least one of the first multimeric protein of interest and the second multimeric protein of interest is a heteromeric protein.

235. The method of any one of claims 217, 221, 225, 226 or 230, wherein at least one of the first protein of interest and the second protein of interest is a dimeric protein.

236. The method of any one of claims 217, 221, 225, 226 or 230, wherein at least one of the first protein of interest and the second protein of interest is a trimeric protein.

237. The method of any one of claims 217, 221, 225, 226 or 230, wherein the first protein of interest and the second protein of interest are different forms of multimeric proteins.

238. The method of claim 237, wherein said multimeric protein is a GABA a receptor.

239. The method of any one of claims 217, 221, 225, 226 or 230, wherein at least one of said first or second protein of interest is part of a functional biological pathway.

240. The method of any one of claims 239, wherein the functional biological pathway is selected from the group consisting of: glycosylation, protein synthesis, UPR, ER, ribosomes, mitochondrial activity, RNA synthesis, post-translational modifications, cell signaling, cell growth, and cell death.

241. The method of any one of claims 217, 221, 225, 226, or 230, wherein the physiological property is a therapeutic effect.

242. The method of any one of claims 217, 221, 225, 226, or 230, wherein the physiological property is an adverse effect.

243. The method of any one of claims 217, 221, 225, 226 or 230, wherein the effect of the compound or compounds on the physiological property is determined using high throughput screening.

244. The method of any one of claims 217, 221, 225, 226 or 230, wherein step e) is performed in a computer system.

245. A computer-implemented method for determining a physiological property of a test compound or a plurality of test compounds, wherein the method comprises:

a. Receiving a first activity profile of the test compound or compounds, wherein the first activity profile is generated by the method of claim 217 or 221, and wherein the first activity profile provides an in vitro correlation of physiological properties of the test compound or compounds;

b. comparing the first activity profile to a plurality of marker activity profiles stored in a database to determine a measure of similarity between the first activity profile and each of the marker activity profiles in the plurality of marker activity profiles, wherein each of the marker activity profiles provides an in vitro correlation of known physiological properties of a respective known compound or a plurality of known compounds;

c. determining one or more signature activity profiles that are most similar to the first activity profile based on the similarity measure determined in step (b):

d. identifying a known physiological property associated with one or more marker activity profiles determined to be most similar to the first activity profile in step (c) as a physiological property of the test compound or compounds;

246. The method of claim 245, wherein the one or more signature activity profiles are most similar to the first activity profile if the similarity measure is above a predetermined threshold.

247. A computer-implemented method for characterizing a test compound or test compounds as being associated with a particular physiological property, wherein the method comprises:

b. clustering a plurality of activity profiles, the plurality of activity profiles comprising the first activity profile and a plurality of signature activity profiles, wherein each of the signature activity profiles provides an in vitro correlation to a known physiological characteristic of a respective known compound or a plurality of known compounds;

c. identifying one or more signature activity profiles of the plurality of signature activity profiles that cluster with the first activity profile; and

d. characterizing a test compound or compounds as being associated with the known physiological property of each known compound or compounds corresponding to one or more marker activity profiles identified in step (c) clustered with the first activity profile;

248. A computer-implemented method of classifying a test compound or a plurality of test compounds by physiological properties using a classifier, wherein the method comprises:

a. training a classifier using a plurality of marker activity profiles stored in a database to classify a test compound or a plurality of test compounds according to a pharmacological property, wherein each of said marker activity profiles provides an in vitro correlation to a known physiological property of a respective known compound or a plurality of known compounds; and

b. processing the first activity profile generated by the method of claim 217 or 221 using the classifier to classify the test compound or compounds according to a physiological property;

249. A computer-implemented method of classifying a test compound or a plurality of test compounds by physiological properties using a classifier, wherein the method comprises:

a. training a classifier using a plurality of signature activity profiles stored in a database to classify a compound or compounds according to pharmacological properties, wherein each of said signature activity profiles provides an in vitro correlation to a known in vivo pharmacological property of the respective compound; and

b. Processing the first activity profile generated by the method of claim 217 or 221 using the classifier to classify the test compound or compounds according to a physiological property, wherein the classifier is trained according to a method comprising:

250. A method for characterizing a combination of active subunits of a multimeric protein of interest in a cell, wherein said method comprises:

a. contacting a first cell expressing a first subunit of a multimeric protein of interest with the test compound or compounds;

b. contacting a second cell expressing a second subunit of the multimeric protein of interest with the test compound or compounds;

c. contacting a third cell expressing the first and second subunits of the multimeric protein of interest with the test compound or compounds;

d. determining the effect of the test compound or compounds on the multimeric protein in a functional assay when the multimeric protein is expressed in a first cell, a second cell and a third cell;

e. inferring whether the first and/or second subunit is part of a biologically active multimeric protein; and

Wherein the features obtained in steps a) to d) provide an in vitro correlation of physiological properties in vivo,

and wherein the first and second subunits of the multimeric protein independently do not comprise a protein tag, are expressed in a cell cultured in the absence of selective pressure, or any combination thereof.

251. The method of claim 250, wherein the multimeric protein of interest is a heterodimer.

252. The method of claim 250, wherein the multimeric protein of interest is a heterotrimer.

253. A method for characterizing a combination of active subunits of a multimeric protein of interest in a cell, wherein said method comprises:

a. contacting a first cell expressing a first subunit of a multimeric protein of interest with a test compound or test compounds;

c. contacting a third cell expressing a third subunit of the multimeric protein of interest with the test compound or test compounds;

d. contacting a fourth cell expressing the first, second and third subunits of the multimeric protein of interest with the test compound or compounds;

e. Determining the effect of the test compound or compounds on the multimeric protein in a functional assay when the multimeric protein of interest is expressed in the first cell, the second cell, the third cell, and the fourth cell;

f. inferring whether the first, second and/or third subunit is part of a biologically active multimeric protein;

wherein the first, second and third subunits of the multimeric protein independently do not comprise a protein tag, are expressed in a cell cultured in the absence of selective pressure or any combination thereof.

254. The method of claim 253, wherein the multimeric protein is a heterotrimer.

255. The method of claim 250, wherein said multimeric protein is a GABA a receptor.

256. A subject panel of cells, wherein the subject panel comprises a first cell and a second cell, wherein the first and second cells have been engineered to express the same subunit of a multimeric protein of interest, wherein the physiological properties of the multimeric protein of interest in the first cell are different from the physiological properties of the multimeric protein in the second cell, and wherein the first and second cells are derived from the same host cell line;

Wherein the subunits of the multimeric protein of interest do not comprise a protein tag, are expressed in a cell cultured in the absence of selective pressure, or any combination thereof.

257. Cloning a panel of cell lines, wherein each cell line has been engineered to express the same subunit of a multimeric protein of interest, and wherein the physiological properties of the multimeric protein in each cell line are different from the physiological properties of the multimeric protein of interest in another cell line of the panel, and wherein the cell lines in the panel of cell lines are derived from the same host cell line;

wherein the subunits of the multimeric protein of interest do not comprise a protein tag, are expressed in cells cultured in the absence of selective pressure, or any combination thereof.

258. The subject panel of claim 257, wherein the subject panel comprises 2 cell lines.

259. The subject panel of claim 257, wherein the subject panel comprises 5 cell lines.

260. The subject panel of claim 257, wherein the subject panel comprises 10 cell lines.

261. The panel of claim 257, wherein the multimeric protein of interest is NaV.

262. Cells engineered to express all of the constituent proteins of a functional biological pathway have been developed.

263. The cell of claim 262, wherein said pathway has at least 5 protein components.

264. The cell of claim 262, wherein said cell is cultured in the absence of selective pressure.

265. The cell of claim 262, wherein a component protein of said biological pathway does not comprise a protein tag.

266. A panel of clonal cell lines comprising a plurality of clonal cell lines, wherein each clonal cell line of the plurality of clonal cell lines is engineered to express a different odorant receptor; wherein the odorant receptor does not comprise a protein tag, or the odorant receptor is consistently and reproducibly produced in a form suitable for use in a functional assay such that the cells have a Z' factor of at least 0.4 in the functional assay, or a clonal cell line is cultured in the absence of selective pressure, or any combination thereof.

267. The subject panel of claim 266, wherein the plurality of clonal cell lines comprises at least 10 cell lines.

268. The subject panel of claim 266, wherein the different odorant receptors are human odorant receptors or insect odorant receptors.

269. The subject group of claim 266, wherein the different human odorant receptors are selected from the group consisting of OR10A, OR10C, OR10D, OR10G, OR10H, OR10J, OR10K, OR10Q, OR10R, OR10S, OR10T, OR10V, OR10Z, OR11A, OR11G, OR11H, OR11L, OR12D, OR13A, OR13C, OR13D, OR13E, OR1J, OR1F 1J, OR2J, OR 1H, OR1J, OR2J, OR 1H, OR1J, OR14, OR1J, OR2A, OR1J, OR14, OR 1H, OR2J, OR14, OR 1H, OR2J, OR14, OR 1H, OR14, OR1J, OR13A, OR13H, OR1J, OR13H, OR13A, OR13C, OR13A, OR2J, OR13A, OR13D, OR13C, OR13D, OR13C, OR13D, OR13A, OR13D, OR13C, OR13D, OR1J, OR1D, OR1J, OR13, OR, OR2B, OR2C, OR2D, OR2F, OR2G, OR2H, OR2J, OR2K, OR2L, OR2M, OR2S, OR2T, OR2V, OR2W, OR2Y, OR2Z, OR3A, OR4B, OR4C, OR4D, OR4K 51, OR4K 51, OR2M, OR2S 52K, OR2S 52, OR2S 51, OR2S 52, OR2T, OR2V, OR2W, OR2Y, OR2Z, OR3A, OR4K, OR4A, OR4K 51, OR4D, OR4A, OR4K 51, OR4D, OR4K 51, OR4, OR51, OR4D, OR4A, OR51, OR4K 51, OR4D, OR4K 51, OR4D, OR4A, OR4D, OR4K 51, OR4D, OR51, OR4K 51, OR4, OR51, OR4D, OR4K 51, OR2S 51, OR4D, OR4, OR51, OR4K 51, OR2S 51, OR4, OR51, OR2S 51, OR4, OR52L, OR52N, OR52P, OR52R, OR56A, OR56B, OR5A, OR5AC, OR5AK, OR5AN, OR5AP, OR5AR, OR5AS, OR5AU, OR5B, OR5C, OR5D, OR5F, OR5G, OR5H, OR5I, OR5K, OR5L, OR5M, OR5P, OR5T, OR6N, OR6A, OR8K, OR6C, OR 7K, OR6K, OR7C, OR6K, OR 7K, OR 6D, OR7C, OR6K, OR 7K, OR6K, OR7C, OR 7K, OR6K, OR 7K, OR 6D, OR 6G 7C, OR 7K, OR 6D, OR5F, OR5G, OR5H, OR6K, OR 7K, OR6K, OR7C, OR6K, OR 7K, OR6K, OR7C, OR 7K, OR.

270. The subject group of claim 266, wherein the different insect scent receptors are mosquito scent receptors selected from IOR100, IOR101, IOR102, IOR103, IOR104, IOR105, IOR106, IOR107, IOR108, IOR109, IOR110, IOR111, IOR112, IOR113, IOR114, IOR115, IOR116, IOR117, IOR118, IOR119, IOR120, IOR121, IOR122, IOR123, IOR124, IOR125, IOR126, IOR127, IOR, 7080, IOR 7180, IOR 7091, IOR 7080, IOR 7097, IOR 7080, ORL7097, ORL7080, IOR 7080, ORL7097, ORL7080, ORL7097, ORL7080, ORL7087, ORL7095, ORL7087, ORL7095, ORL7080, ORL7087, ORL7080, ORL7095, ORL7087, ORL7095, ORL7087, ORL7095, ORL7087, TPR 7108, ORL7109, ORL7110, ORL7111, ORL7112, ORL7113, ORL7114, ORL7115, ORL7116, ORL7117, ORL7118, ORL7119, ORL7120, ORL7121, ORL7122, ORL7123, ORL7124, ORL7125, TPR2307, TPR2308, TPR2309, TPR2310, TPR2312, TPR2314, TPR2315, TPR2316, TPR2317, TPR2318, TPR2319, TPR2320, TPR772 1, TPR2321, TPR698 735, TPR699, TPR700, TPR701, TPR702, TPR703, TPR704, TPR705, 722, 706, TPR709, TPR711, TPR 320520, TPR769, TPR 7646, TPR769, TPR 73471070, TPR 73479, TPR769, TPR 73479, TPR 734773479, TPR 73479, TPR 732049, TPR769, TPR 732049, TPR769, TPR 732049, TPR769, TPR 7646, TPR 732049, TPR769, TPR 732049, TPR769, TPR 7646, TPR 732049, TPR769, TPR.

271. A method for generating an odor activity profile of a test compound or composition, wherein the method comprises:

a. contacting the subject group of claim 266 with the test compound or composition; and

b. measuring the effect of said test compound or composition on the activity of at least 2 different odorant receptors in a functional test in a group of subjects,

wherein the activity measured in step (b) provides an odor activity profile of the test compound or composition.

272. A method for identifying a second test compound that mimics the odor of a first test compound or composition, wherein the method comprises:

a. contacting the subject group of claim 266 with the second test compound;

b. testing the effect of the second test compound on the activity of at least 2 odorant receptors in a group of subjects in a functional assay;

c. comparing the odor activity spectrum of the second test compound obtained in step (b) with the odor activity spectrum of the first test compound or composition; wherein the second test compound mimics the odor of the first test compound or composition if the odor activity spectrum of the second test compound is similar to the odor activity spectrum of the first test compound or composition.

273. A method for identifying a second test compound that alters the odor activity profile of a first test compound or composition, wherein the method comprises:

a. generating an odor activity profile of a second test compound in the presence of the first test compound or composition according to the method of claim 271;

b. comparing the odor activity profile obtained in step (a) with the odor activity profile of the first test compound or composition in the absence of the second test compound; wherein the second test compound alters the odor activity spectrum of the first test compound or composition if the odor activity spectrum of the first test compound or composition alone is different from the odor activity spectrum of the second test compound in the presence of the first test compound or composition.

274. A computer-implemented method for identifying an odor associated with a test compound, wherein the method comprises:

a. receiving a first odor activity profile of a test compound, wherein the first odor activity profile is produced by the method of claim 271;

b. comparing the first odor activity spectrum to a plurality of hallmark odor activity spectra stored in a database to determine a measure of similarity between the first odor activity spectrum and each of the hallmark odor activity spectra in the plurality of hallmark odor activity spectra, wherein each of the hallmark odor activity spectra corresponds to a respective known compound having a known odor, and wherein each of the hallmark odor activity spectra was generated by the method of claim 271;

c. Determining one or more signature odor activity profiles that are most similar to the first odor activity profile based on the similarity measure determined in step (b); and

d. identifying an odor associated with one or more hallmark odor activity profiles determined to be most similar to the first odor activity profile in step (c) as an odor associated with the known compound;

275. The method of claim 274, wherein the similarity measure is above a predetermined threshold, then the one or more hallmark odor activity profiles are most similar to the first odor activity profile.

276. A computer-implemented method for characterizing a compound as being associated with a particular odor, wherein the method comprises:

a. receiving a first odor activity profile of the compound, wherein the first odor activity profile is produced by the method of claim 271;

b. clustering a plurality of odor activity profiles, the plurality of odor activity profiles comprising the first odor activity profile and a plurality of hallmark odor activity profiles, wherein each of the hallmark odor activity profiles corresponds to a respective known compound having a known odor, and wherein the hallmark odor activity profiles are generated by the method of claim 271;

c. Identifying one or more hallmark odor activity profiles of the plurality of hallmark odor activity profiles clustered with a first odor activity profile; and

d. characterizing compounds as being associated with the known odors associated with each compound corresponding to one or more hallmark odor activity profiles identified in step (c) as clustered with the first odor activity profile;

277. A computer-implemented method of classifying a test compound as having an odor using a classifier, wherein the method comprises:

a. training a classifier using a plurality of signature odor activity profiles stored in a database to classify test compounds according to odor, wherein each of the signature odor activity profiles corresponds to a known compound each having a known odor, and wherein each of the signature odor activity profiles is generated by the method of claim 271; and

b. processing a first odor activity spectrum of the compound produced by the method of claim 271 with the classifier to classify the compound by known odor;

278. A computer-implemented method of classifying a test compound as having an odor using a classifier, wherein the method comprises:

a. processing a first odor activity spectrum of the compound produced by the method of claim 271 with the classifier to classify the compound by known odor, wherein the classifier is trained according to a method that:

b. training a classifier using a plurality of signature odor activity profiles stored in a database to classify test compounds according to odor, wherein each of the signature odor activity profiles corresponds to a known compound each having a known odor, and wherein each of the signature odor activity profiles is generated by the method of claim 271;

where the processing is performed on a computer using suitable programming.

279. A computer-implemented method for correlating one or more test compounds with odor, wherein the method comprises:

a. a first odor activity profile of a test compound that accepts a first test compound, wherein the first odor activity profile is produced by the method of claim 271, and wherein the first test compound has a known odor;

b. comparing the first odor activity profile to a plurality of hallmark odor activity profiles stored in a database to determine a measure of similarity between the first odor activity profile and each of the hallmark odor activity profiles in the plurality of hallmark odor activity profiles, wherein each of the hallmark odor activity profiles corresponds to a respective known compound, and wherein each of the hallmark odor activity profiles is generated by the method of claim 271;

c. Determining one or more hallmark odor activity profiles that are most similar to the first odor activity profile based on the similarity measure determined in step (b); and

d. characterizing each test compound corresponding to the one or more hallmark odor activity profiles determined to be most similar to the first odor activity profile in step (c) as being associated with the known odor;

280. The method of claim 279, wherein the one or more hallmark odor activity profiles are most similar to the first odor activity profile if the similarity measure is above a predetermined threshold.

281. A computer-implemented method for characterizing one or more test compounds as being associated with a particular odor, wherein the method comprises:

a. accepting a first odor activity profile of a first test compound, wherein the first odor activity profile is produced by the method of claim 271 and the first test compound has a known odor;

b. clustering a plurality of odor activity profiles, the plurality of odor activity profiles comprising the first odor activity profile and a plurality of hallmark odor activity profiles, wherein each of the hallmark odor activity profiles corresponds to a respective known compound, and wherein each of the hallmark odor activity profiles is generated by the method of claim 271;

d. characterizing each compound corresponding to one or more hallmark odor activity profiles identified in step (c) as clustered with the first odor activity profile as being associated with the known odor;

282. A computer-implemented method of classifying one or more test compounds as having an odor using a classifier, wherein the method comprises:

a. processing a first odor activity profile generated by the method of claim 271 with the classifier, wherein the first odor activity profile corresponds to a first test compound having a known odor to classify one or more signature odor activity profiles of a plurality of signature odor activity profiles stored in a database as having the known odor, wherein the classifier is trained in accordance with a method comprising:

b. training the classifier to classify the one or more signature odor activity profiles as having an odor using the plurality of signature odor activity profiles, wherein each of the signature odor activity profiles corresponds to a respective known compound, and wherein each of the signature odor activity profiles is generated by the method of claim 271;

Where the processing is performed on a computer using suitable programming.

283. A protein or proteins that associate in vitro with a protein or proteins of interest in vivo, wherein the in vitro association is predictive of the function or activity of the corresponding protein or proteins of interest expressed in vivo; wherein the in vitro correlation is a biologically active protein or proteins expressed under in vitro non-physiological conditions; wherein the in vitro correlation comprises at least one functional or pharmacological or physiological property corresponding to the protein or proteins of interest in vivo; and wherein at least 10% of the compounds identified in the high throughput screening using the in vitro correlation are capable of having an in vivo therapeutic effect.

284. The protein of claim 283, wherein the in vitro correlation comprises at least 2, 3, 4, 5, or 6 subunits.

285. The plurality of proteins of claim 283, wherein at least one protein of said in vitro correlation comprises at least 2, 3, 4, 5, or 6 subunits.

286. The protein of claim 283, wherein the in vitro correlation comprises a heteromultimer.

287. The plurality of proteins of claim 283, wherein at least one protein of said in vitro correlation comprises a heteromultimer.

288. The protein or proteins of any one of claims 283-287, wherein the in vitro related protein or proteins do not comprise a protein tag.

289. The protein or proteins of any one of claims 283-288, wherein said in vitro correlation is stably expressed in cells cultured in the absence of selective pressure.

290. The protein or proteins of any one of claims 283-289, wherein the in vitro correlation is expressed in a cell line without causing cytotoxicity.

291. The protein or proteins of any one of claims 283-290, wherein the in vitro correlation is expressed in a cell that does not endogenously express the protein or proteins.

292. A cell expressing the protein or proteins of any one of claims 283-291.

293. A cell line produced from the cell of claim 292.

294. A method for identifying a modulator of a protein of interest in vivo, said method comprising the steps of:

a. contacting the cell of claim 292 with a test compound; and

b. detecting a change in activity of the in vitro relevant protein or proteins in cells contacted with the test compound as compared to the activity of the in vitro relevant protein or proteins in cells not contacted with the test compound;

295. A modulator identified by the method of claim 294.

296. The cell of any one of claims 190, 192, 194, 195-199, 201-202, 262, and 292, wherein said cell is a differentiated cell.

297. The cell of any one of claims 190, 192, 194, 195-199, 201-202, 262, and 292 wherein said cell is a dedifferentiated cell.

298. The cell of claim 297, wherein the dedifferentiated cell is a stem cell selected from the group consisting of: pluripotent stem cells, multipotent stem cells, totipotent stem cells, induced pluripotent stem cells, embryonic stem cells, cancer stem cells, organ-specific stem cells, and tissue-specific stem cells.

299. A method of producing stem cells, comprising the steps of: dedifferentiating the differentiated cells into stem cells, wherein the differentiated cells are the cells of claim 296.

300. A method for producing redifferentiated cells, comprising the steps of:

a. dedifferentiating the stem cells of claim 296 to produce stem cells; and

b. Redifferentiating the stem cells to produce redifferentiated cells.

301. The method of claim 299 or claim 300, wherein the stem cell is selected from the group consisting of: pluripotent stem cells, multipotent stem cells, totipotent stem cells, induced pluripotent stem cells, embryonic stem cells, cancer stem cells, organ-specific stem cells, and tissue-specific stem cells.

302. The method of claim 300, wherein the redifferentiated cells are of a different type than the cells of claim 296.

303. A method for producing a non-human organism, comprising the steps of:

a. dedifferentiating the cells of claim 296 to produce stem cells, wherein the stem cells are embryonic stem cells or induced pluripotent stem cells; and

b. redifferentiating the cells to produce a non-human organism.

304. The method of claim 303, wherein the organism is a mammal.

305. The method of claim 304, wherein the mammal is a mouse.

306. A redifferentiated cell produced by the method of claim 300.

307. A non-human organism produced by the method of claim 303.

308. The non-human organism of claim 307, wherein said organism is a mammal.

309. The non-human organism of claim 308, wherein said mammal is a mouse.

310. The method of claim 218 or claim 227, wherein the first and second proteins of interest are independently selected from the group consisting of: ENaC, NaV, GABAA, sweet taste receptor, umami taste receptor, bitter taste receptor, CFTR and GCC.

311. The method of claim 231, wherein each protein is independently selected from the group consisting of: ENaC, NaV, GABAA, sweet taste receptor, umami taste receptor, bitter taste receptor, CFTR and GCC.

Claims

a. providing a population of cells;

6. An isolated cell produced according to any one of claims 1 to 5.

8. The cell of claim 7, wherein the population of cells is a population of Chinese Hamster Ovary (CHO) cells, an established neuronal cell line, pheochromocytoma, neuroblastoma fibroblasts, rhabdomyosarcoma cells, dorsal root ganglion cells, NS0 cells, CV-1(ATCC CCL 70), COS-1(ATCC CRL 1650), COS-7(ATCC CRL 1651), CHO-K1(ATCC CCL61), 3T3(ATCC CCL 92), NIH/3T3(ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1(ATCC CCL 26), C-5(ATCC CCL 171), L-cells, HEK-293(ATCC CRL1573), and PC12(ATCC CRL-1721), HEK293T (ATCC CRL-11268), RBL-SY 8), SH-2265 (cccrl-5Y) (ATCC CRL 1376-1376), ATCC CRL 1721 (ATCC CRL 1378), and ATCC CRL 1376 (ATCC CRL 1376), MDCK (ATCC CCL-34), SJ-RH30(ATCC CRL-2061), HepG2(ATCC HB-8065), ND7/23(ECACC 92090903), CHO (ECACC85050302), Vero (ATCC CCL 81), Caco-2(ATCC HTB 37), K562(ATCC CCL243), Jurkat (ATCC TIB-152), Per.C6(Crucell, Leiden, The Netherlands), Huvec (ATCC human primary PCS 100-type 010, mouse CRL 2514, CRL 2515, CRL 2516), HuH-7D12 (ECTB 01042712), 293(ATCC CRL 10852), A549(ATCC CCL185), IMR-90(ATCC CCL OS), MCF-7(ATC HTB-22), CHU-2 (CHTB-96), T84(ATCC CCL 248), primary ACC 248 or immortalized cells.

9. A culture of the cell of claim 7.

14. The cell or cell line of claim 10 or 11, wherein the host cell:

a. is a eukaryotic cell;

b. is a mammalian cell;

Any combination of (a), (b), and (c).

16. The cell or cell line of claim 10 or 11, wherein the sweet taste receptor

a. Is a mammalian sweet taste receptor;

b. is a human sweet receptor;

c. comprise subunits from different species;

d. comprises one or more subunits that are chimeric;

any combination of (a) - (d).

a. comprises the amino acid sequence of SEQ ID NO: 31;

a. Comprises the amino acid sequence of SEQ ID NO: 32;

a. comprises the amino acid sequence of SEQ ID NO: 33;

29. The method of claim 27, wherein the host cell:

a. is a eukaryotic cell;

b. is a mammalian cell;

d.a), b) and c).

30. The method of claim 27, further comprising the steps of:

31. The method of claim 27, further comprising the steps of:

37. The method of claim 35, wherein the modulator is selected from the group consisting of a sweet taste receptor inhibitor, a sweet taste receptor antagonist, a sweet taste receptor blocker, a sweet taste receptor activator, a sweet taste receptor agonist, or a sweet taste receptor enhancer.

38. The method of claim 35, wherein the sweet taste receptor is a human sweet taste receptor.

39. The method of claim 35, wherein the test compound is a small molecule, chemical moiety, polypeptide, or antibody.

40. The method of claim 35, wherein the test compound is a library of compounds.

42. The method of claim 35, wherein the modulator is selective for enzymatically modified forms of sweet taste receptors.

43. A modulator identified by the method of claim 35.

54. The cell or cell line of claim 50 or 51, wherein the host cell:

a. is a eukaryotic cell;

b. is a mammalian cell;

Any combination of (a), (b), and (c).

56. The cell or cell line of claim 50 or 51, wherein the umami receptor

a. Is a mammal;

b. is a human;

c. comprise subunits from different species;

d. comprises one or more subunits that are chimeric;

any combination of (a) - (d).

c. comprising a polypeptide consisting of a sequence that hybridizes under stringent conditions to SEQ ID NO: 41 or encodes a polypeptide selected from the group consisting of SEQ ID NOs: 42-45 or a nucleic acid that hybridizes to a nucleic acid of a corresponding amino acid sequence of another species; and

a. comprises the amino acid sequence of SEQ ID NO: 41;

c. A nucleic acid comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid of a) or b); comprises a sequence identical to SEQ ID NO: 41 or encodes a polypeptide selected from SEQ ID NO: 42-45 or a nucleic acid of a nucleotide sequence that is at least 95% identical to a nucleic acid of a corresponding amino acid sequence of another species.

a. comprises the amino acid sequence of SEQ ID NO: 32;

a. comprises the amino acid sequence of SEQ ID NO: 33 or another species;

68. The method of claim [0207], further comprising the step of generating a cell line from the cells isolated in step c).

69. The method of claim [0207], wherein said host cell:

a. is a eukaryotic cell;

b. is a mammalian cell;

d.a), b) and c).

70. The method of claim [0207], further comprising the steps of:

71. The method of claim [0207], further comprising the steps of:

73. The method of claim [0207], wherein the T1R1 subunit is selected from the subunits of claims 12a) to d), wherein the T1R3 subunit is selected from the subunits of claims 14a) to d) and wherein the G protein is selected from the proteins of claims 16a) to d).

74. The method of claim [0207], wherein the T1R1 subunit encoded by the nucleic acid is selected from the group of subunits encoded by the nucleic acids of claims 13a) to d), wherein the T1R3 subunit is selected from the group of subunits encoded by the nucleic acids of claims 15a) to d) and wherein the G protein is selected from the group of proteins encoded by the nucleic acids of claims 17a) to d).

75. The method of claim [0207], wherein said separating step utilizes a fluorescence activated cell sorter.

77. The method of claim [0213], wherein the modulator is selected from an umami receptor inhibitor, an umami receptor antagonist, an umami receptor blocker, an umami receptor activator, an umami receptor agonist, or an umami receptor potentiator.

78. The method of claim [0213], wherein the umami receptor is a human umami receptor.

79. The method of claim [0213], wherein the test compound is a small molecule, chemical moiety, polypeptide, or antibody.

80. The method of claim [0213], wherein the test compound is a library of compounds.

82. The method of claim [0213], wherein the modulator is selective for an enzymatically modified form of the umami receptor.

83. A modulator identified by the molecule of claim [0213 ].

90. A cell or cell line engineered to stably express a bitter taste receptor.

98. The cell or cell line of claim 90, wherein the cell or cell line stably expresses: to stably express bitter taste receptors.

a. An endogenous G protein;

b. a heterologous G protein or

c. And both.

a. A eukaryotic cell;

b. a mammalian cell;

c. cells that do not express endogenous bitter taste receptors; or

Any combination of (a), (b), and (c).

103. The cell or cell line of claim 90, wherein the bitter taste receptor:

a. is a mammal;

b. is a human;

any combination of (a), (b), and (c).

seq ID NOS: any one of 77-101;

seq ID NOS: any one of 51-75;

d. as SEQ ID NOS: 51-75, or a variant thereof.

116. The collection of claim 114 wherein the allelic variants thereof are SNPs.

126. The collection of claim 124 wherein the allelic variants thereof are SNPs.

c. isolating cells expressing the bitter taste receptor.

137. The method of claim 134, wherein the cell is:

a. a eukaryotic cell;

b. a mammalian cell;

c. cells that do not express endogenous bitter taste receptors; or

Any combination of (a), (b), and (c).

139. The method of claim 134, wherein the bitter taste receptor:

a. is a mammal;

b. is a human;

any combination of (a), (b), and (c).

Seq ID NOS: any one of 77-101;

seq ID NOS: any one of 51-75;

d. as SEQ ID NOS: 51-75, or a variant thereof.

a. prior to introducing the nucleic acid encoding the bitter taste receptor;

b. after introducing the nucleic acid encoding the bitter taste receptor; or

151. The method of claim 58, further comprising:

a. prior to isolating cells expressing the bitter taste receptor;

b. after isolating cells expressing the bitter taste receptor; or

b. detecting a change in function of the bitter taste receptor.

156. The method of claim 152, wherein said cell or a cell in said cell line is:

a. a eukaryotic cell;

b. a mammalian cell;

c. cells that do not express any endogenous bitter taste receptors; or

Any combination of (a), (b), and (c).

158. The method of claim 152, wherein the bitter taste receptor:

a. is a mammal;

b. is a human;

Any combination of (a), (b), and (c).

seq ID NOS: any one of 77-101;

Seq ID NOS: any one of 51-75;

183. The matched panel of clonal cell lines of claim [0079], wherein the cell lines are selected from primary cells and immortalized cells.

184. The matched panel of clonal cell lines of claim [0079], wherein the clonal cell line cells are eukaryotic cells and are selected from the group consisting of: fungal cells, insect cells, mammalian cells, yeast cells, algae, crustacean cells, arthropod cells, avian cells, reptilian cells, amphibian cells, plant cells, humans, non-human primates, bovines, porcines, felines, rats, marsupials, murines, canines, ovines, caprines, rabbits, guinea pigs, hamsters.

185. The matched panel of clonal cell lines of claim [0079], wherein cells in the cell line are engineered to express the protein of interest.

186. The matched panel of clonal cell lines of claim [0079], wherein cells in the cell line express the protein of interest from an introduced nucleic acid encoding the protein or, in the case of a multimeric protein, a protein subunit.

187. The matched panel of clonal cell lines of claim [0079], wherein the cells express the protein of interest from an endogenous nucleic acid and wherein the cells are engineered to activate transcription of an endogenous protein, or in the case of a multimeric protein, of a protein subunit.

188. The matched panel of clonal cell lines of claim [0079], wherein the panel comprises at least 4, at least 6, or at least 20 clonal cell lines.

189. The matched panel of clonal cell lines of claim [0079], wherein the multi-subunit protein is selected from the group consisting of: ion channels, G protein-coupled receptors (GPCRs), tyrosine receptor kinases, cytokine receptors, nuclear steroid hormone receptors, antibodies, biologicals, and immunoreceptors.

200. The cell of any one of claims [0100] - [0104], wherein said cell is produced in a time period selected from the group consisting of less than 1 week, less than 2 weeks, less than 3 weeks, less than 4 weeks, less than 1 month, less than 2 months, less than 3 months, less than 4 months, less than 5 months, less than 6 months, less than 7 months, less than 8 months, or less than 9 months.

203. The cell of claim 168 or 169, wherein said cell is produced in a time period selected from less than 3 months, less than 4 months, or less than 6 months.

204. The cell of any one of claims [0100] -203, wherein said protein is a monomeric protein.

205. The cell of any one of claims [0100] -203, wherein said protein is a multimeric protein.

206. The cell of any one of claims [0100] -203, wherein said protein of interest does not comprise a protein tag or said cell is cultured in the absence of selective pressure, or a combination thereof.

210. A cell line produced from the cell of any one of claims [0100] -209.

a. contacting a cell of any one of claims [0100] to 210 with a test compound; and

212. A matched panel of cells or clonal cell lines comprising at least 2 cells according to claims [0100] -209 or 2 clonal cell lines of claim 210, wherein said at least 2 cells or said at least 2 clonal cell lines are matched such that they are cultured in parallel under the same cell culture conditions.

213. The matched panel of claim [0111], wherein the matched panel comprises at least 10 cells of claims [0100] -209 or 10 clonal cell lines of claim 210, and the at least 10 cells or 10 clonal cell lines are matched such that they are cultured in parallel under the same cell culture conditions.

214. The matched panel of claim 213, wherein the panel comprises at least 100 cells of claims [0100] -209 or at least 100 of claims 210, and the at least 100 cells or the at least 100 clonal cell lines are cultured in parallel under the same cell culture conditions.

a. Said group of subjects comprises at least 5 cell lines;

b. generating the subject group in less than 6 months;

c. the first and second proteins of interest do not have a protein tag;

d. culturing the clonal cell line in the absence of selective pressure; or

Any combination of e.a) -d).

216. The matched panel of claim [0113], wherein the first protein of interest is an antibody and the functional biological pathway is a glycosylation pathway.

218. The method of claim [0115], wherein the first and second proteins of interest are independently selected from monomeric or multimeric proteins.

221. The method of any one of claims [0115] -219, wherein:

a. contacting the test compound or compounds with a first cell expressing a first protein of interest according to claim [0115] -220;

c. contacting a test compound or a plurality of said test compounds with a second cell expressing a second protein of interest according to claim [0115] -220;

e. comparing the activity profile of the test compound obtained in steps a) to d) with the in vitro correlation as generated by the method of claim [0115 ].

a. contacting the test compound or compounds with a first cell expressing a first protein of interest according to claim [0115 ];

c. contacting the test compound or compounds with a second cell expressing a second protein of interest according to claim [0115 ];

e. comparing the activity profile of the test compound or compounds obtained in steps a) to d) with the in vitro correlation of the physiological property as produced by the method of claim [0115],

227. The method of any one of claims [0119] - [0121], wherein the first and second proteins are independently selected from monomeric or multimeric proteins.

230. The method of any one of claims [0119] -229, wherein:

234. The method of any one of claims [0115], 221, [0119], [0121], or 230, wherein at least one of the first and second multimeric proteins of interest is a heteromeric protein.

235. The method of any one of claims [0115], 221, [0119], [0121], or 230, wherein at least one of the first protein of interest and the second protein of interest is a dimeric protein.

236. The method of any one of claims [0115], 221, [0119], [0121], or 230, wherein at least one of the first protein of interest and the second protein of interest is a trimeric protein.

237. The method of any one of claims [0115], 221, [0119], [0121], or 230, wherein the first protein of interest and the second protein of interest are different forms of a multimeric protein.

239. The method of any one of claims [0115], 221, [0119], [0121], or 230, wherein at least one of said first or second protein of interest is part of a functional biological pathway.

241. The method of any one of claims [0115], 221, [0119], [0121], or 230, wherein the physiological property is a therapeutic effect.

242. The method of any one of claims [0115], 221, [0119], [0121], or 230, wherein the physiological property is an adverse effect.

243. The method of any one of claims [0115], 221, [0119], [0121], or 230, wherein the effect of the compound or compounds on the physiological property is determined using a high throughput screen.

244. The method of any one of claims [0115], 221, [0119], [0121], or 230, wherein step e) is performed in a computer system.

a. Receiving a first activity profile of the test compound or compounds, wherein the first activity profile is generated by the method of claim [0115] or 221, and wherein the first activity profile provides an in vitro correlation of physiological properties of the test compound or compounds;

246. The method of claim [0127], wherein said one or more signature activity profiles are most similar to said first activity profile if said similarity measure is above a predetermined threshold.

b. processing the first activity profile generated by the method of claim [0115] or 221 using the classifier to classify the test compound or compounds according to a physiological property;

b. Processing the first activity profile generated by the method of claim [0115] or 221 using the classifier to classify the test compound or compounds according to a physiological characteristic, wherein the classifier is trained according to a method comprising:

251. The method of claim [0132], wherein the multimeric protein of interest is a heterodimer.

252. The method of claim [0132], wherein the multimeric protein of interest is a heterotrimer.

254. The method of claim [0134], wherein the multimeric protein is a heterotrimer.

255. The method of claim [0132], wherein the multimeric protein is a GABAA receptor.

258. The panel of claim [0137], wherein the panel comprises 2 cell lines.

259. The panel of claim [0137], wherein the panel comprises 5 cell lines.

260. The panel of claim [0137], wherein the panel comprises 10 cell lines.

261. The panel of claim [0137], wherein the multimeric protein of interest is NaV.

263. The cell of claim [0140], wherein the pathway has at least 5 protein components.

264. The cell of claim [0140], wherein the cell is cultured in the absence of selective pressure.

265. The cell of claim [0140], wherein the constituent proteins of the biological pathway do not comprise a protein tag.

267. The panel of claim [0142], wherein the plurality of clonal cell lines includes at least 10 cell lines.

268. The panel of claim [0142], wherein the different odorant receptors are human odorant receptors or insect odorant receptors.

269. The subject group of [0142], wherein the different human odorant receptors are selected from OR10A, OR10C, OR10D, OR10G, OR10H, OR10J, OR10K, OR10Q, OR10R, OR10S, OR10T, OR10V, OR10Z, OR11A, OR11G, OR11H, OR11L, OR12D, OR13A, OR13C, OR13D, OR1E 1J, OR 1H, OR1J, OR2J, OR1F 14J, OR1A, OR14J, OR1J, OR14, OR1J, OR2D, OR 1H, OR1J, OR14, OR 1H, OR14, OR1J, OR 1H, OR14, OR1J, OR14, OR 1H, OR14, OR 1H, OR14, OR 1H, OR1J, OR14, OR1J, OR 1H, OR14, OR 1H, OR14A, OR1D, OR14A, OR1D, OR2D, OR 1H, OR14, OR 1H, OR13A, OR 1H, OR2D, OR14, OR1D, OR14, OR1A, OR14, OR 1H, OR14, OR1D, OR2A, OR1, OR2B, OR2C, OR2D, OR2F, OR2G, OR2H, OR2J, OR2K, OR2L, OR2M, OR2S, OR2T, OR2V, OR2W, OR2Y, OR2Z, OR3A, OR4B, OR4C, OR2D, OR2G, OR2H, OR2T, OR2V, OR2W, OR2Y, OR2Z, OR4C 51, OR4A, OR4K, OR4A, OR4K 51, OR4D, OR4K, OR4A, OR4K 51, OR4D, OR4K, OR4D, OR4K 51, OR4D, OR4K, OR51, OR2S 51, OR2W, OR4K, OR2S 51, OR4K 51, OR2S 51, OR4K, OR4A, OR4K 51, OR4K, OR51, OR4K 51, OR4D, OR4A, OR4D, OR51, OR4K, OR51, OR4D, OR4A, OR4D, OR4K, OR4D, OR51, OR4D, OR4K, OR4, OR51, OR4A, OR4, OR51, OR4K, OR4, OR51, OR4D, OR4K, OR52L, OR52N, OR52P, OR52R, OR56A, OR56B, OR5A, OR5AC, OR5AK, OR5AN, OR5AP, OR5AR, OR5AS, OR5AU, OR5B, OR5C, OR5D, OR5F, OR5G, OR5H, OR5I, OR5K, OR5L, OR5M, OR5P, OR5T, OR 5N, OR6K, OR 7K, OR6A, OR 7K, OR6K, OR7C, OR7D, OR 7K, OR6K, OR7C, OR7D, OR6K, OR 7K, OR 6D, OR5F, OR5H, OR5I, OR5K, OR6K, OR 7K, OR6K, OR 7K, OR6K, OR 7K, OR6K, OR7C, OR7, OR 6K.

270. The subject group of claim [0142], wherein the different insect odorant receptors are mosquito odorant receptors selected from IOR100, IOR101, IOR102, IOR103, IOR104, IOR105, IOR106, IOR107, IOR108, IOR109, IOR110, IOR111, IOR112, IOR113, IOR114, IOR115, IOR116, IOR117, IOR118, IOR119, IOR120, IOR121, IOR122, IOR123, IOR124, IOR125, IOR126, IOR127, IOR 7180, IOR 7091, IOR 7080, IOR 7091, IOR 7081, IOR 7080, IOR 7091, IOR 7080, IOR 7097, ORL7080, IOR 7097, ORL7080, IOR 7097, IOR 7080, ORL7097, IOR 7080, IOR 7097, IOR 7087, IOR 7080, ORL7087, IOR 7080, IOR 7097, IOR 7080, IOR 7087, IOR 7080, IOR 7095, IOR 7080, IOR 7095, IOR 7080, IOR70, TPR 7108, ORL7109, ORL7110, ORL7111, ORL7112, ORL7113, ORL7114, ORL7115, ORL7116, ORL7117, ORL7118, ORL7119, ORL7120, ORL7121, ORL7122, ORL7123, ORL7124, ORL7125, TPR2307, TPR2308, TPR2309, TPR2310, TPR2312, TPR2314, TPR2315, TPR2316, TPR2317, TPR2318, TPR2319, TPR2320, TPR772 1, TPR2321, TPR698 735, TPR699, TPR700, TPR701, TPR702, TPR703, TPR704, TPR705, 722, 706, TPR709, TPR711, TPR 320520, TPR769, TPR 7646, TPR769, TPR 73471070, TPR 73479, TPR769, TPR 73479, TPR 734773479, TPR 73479, TPR 732049, TPR769, TPR 732049, TPR769, TPR 732049, TPR769, TPR 7646, TPR 732049, TPR769, TPR 732049, TPR769, TPR 7646, TPR 732049, TPR769, TPR.

a. contacting the subject group of claim [0142] with the test compound or composition; and

a. contacting the subject group of claim [0142] with the second test compound;

a. generating an odor activity profile of a second test compound in the presence of the first test compound or composition according to the method of claim [0146 ];

a. receiving a first taste activity profile of a test compound, wherein the first taste activity profile is produced by the method of claim [0146 ];

b. comparing the first odor activity profile to a plurality of hallmark odor activity profiles stored in a database to determine a measure of similarity between the first odor activity profile and each of the hallmark odor activity profiles in the plurality of hallmark odor activity profiles, wherein each of the hallmark odor activity profiles corresponds to a respective known compound having a known odor, and wherein each of the hallmark odor activity profiles is generated by the method of claim [0146 ];

275. The method of claim [0149], wherein the similarity measure is above a predetermined threshold, then the one or more hallmark odor activity profiles are most similar to the first odor activity profile.

276. A computer-implemented method for characterizing a compound as being associated with a particular odor, wherein the method comprises: a. receiving a first taste activity profile of said compound, wherein said first taste activity profile is produced by the method of claim [0146 ];

b. clustering a plurality of odor activity profiles, the plurality of odor activity profiles comprising the first odor activity profile and a plurality of hallmark odor activity profiles, wherein each of the hallmark odor activity profiles corresponds to a respective known compound having a known odor, and wherein the hallmark odor activity profiles are generated by the method of claim [0146 ];

a. training a classifier using a plurality of hallmark odor activity profiles stored in a database to classify test compounds according to odor, wherein each of the hallmark odor activity profiles corresponds to a known compound each having a known odor, and wherein each of the hallmark odor activity profiles is generated by the method of claim [0146 ]; and

b. processing a first odor activity spectrum of the compound produced by the method of claim [0146] using the classifier to classify the compound according to known odor;

a. processing a first odor activity profile of the compound produced by the method of claim [0146] using the classifier to classify the compound according to known odor, wherein the classifier is trained according to a method that:

b. training a classifier using a plurality of hallmark odor activity profiles stored in a database to classify test compounds according to odor, wherein each of the hallmark odor activity profiles corresponds to a known compound each having a known odor, and wherein each of the hallmark odor activity profiles is generated by the method of claim [0146 ];

where the processing is performed on a computer using suitable programming.

a. a first odor activity profile of a test compound that accepts a first test compound, wherein the first odor activity profile is produced by the method of claim [0146], and wherein the first test compound has a known odor;

b. Comparing the first odor activity profile to a plurality of hallmark odor activity profiles stored in a database to determine a measure of similarity between the first odor activity profile and each of the hallmark odor activity profiles in the plurality of hallmark odor activity profiles, wherein each of the hallmark odor activity profiles corresponds to a respective known compound, and wherein each of the hallmark odor activity profiles is generated by the method of claim [0146 ];

280. The method of claim [0161], wherein the one or more hallmark odor activity profiles are most similar to the first odor activity profile if the similarity measure is above a predetermined threshold.

a. Receiving a first odor activity profile of a first test compound, wherein the first odor activity profile is produced by the method of claim [0146] and the first test compound has a known odor;

b. clustering a plurality of odor activity profiles, the plurality of odor activity profiles comprising the first odor activity profile and a plurality of hallmark odor activity profiles, wherein each of the hallmark odor activity profiles corresponds to a respective known compound, and wherein each of the hallmark odor activity profiles is generated by the method of claim [0146 ];

a. processing a first odor activity profile generated by the method of claim [0146] with the classifier, wherein the first odor activity profile corresponds to a first test compound having a known odor, to classify one or more signature odor activity profiles of a plurality of signature odor activity profiles stored in a database as having the known odor, wherein the classifier is trained in accordance with a method comprising:

b. Training the classifier using the plurality of hallmark odor activity profiles to classify the one or more hallmark odor activity profiles as having an odor, wherein each of the hallmark odor activity profiles corresponds to a respective known compound, and wherein each of the hallmark odor activity profiles is generated by the method of claim [0146 ];

where the processing is performed on a computer using suitable programming.

284. The protein of claim [0175], wherein the in vitro correlation comprises at least 2, 3, 4, 5, or 6 subunits.

285. The plurality of proteins of claim [0175], wherein the at least one protein of in vitro correlation comprises at least 2, 3, 4, 5, or 6 subunits.

286. The protein of claim [0175], wherein the in vitro correlation comprises a heteromultimer.

287. The plurality of proteins of claim [0175], wherein the at least one protein of in vitro correlation comprises a heteromultimer.

288. The protein or proteins of any one of claims [0175] -287, wherein the in vitro related protein or proteins do not comprise a protein tag.

289. The protein or proteins of any one of claims [0175] -288, wherein said in vitro correlation is stably expressed in cells cultured in the absence of selective pressure.

290. The protein or proteins of any one of claims [0175] -289, wherein the in vitro correlation is expressed in a cell line without causing cytotoxicity.

291. The protein or proteins of any one of claims [0175] -290, wherein the in vitro correlation is expressed in a cell that does not endogenously express the protein or proteins.

292. A cell expressing the protein or proteins of any one of claims [0175] -291.

293. A cell line produced from the cell of claim 292.

a. contacting the cell of claim 292 with a test compound; and

295. A modulator identified by the method of claim 294.

296. The cell of any one of claims 190, 192, [0097], [0100] - [0104], 201-, [0140] and 292, wherein the cell is a differentiated cell.

297. The cell of any one of claims 190, 192, [0097], [0100] - [0104], 201-oz 202, [0140] and 292, wherein the cell is a dedifferentiated cell.

300. A method for producing redifferentiated cells, comprising the steps of:

a. dedifferentiating the stem cells of claim 296 to produce stem cells; and

b. redifferentiating the stem cells to produce redifferentiated cells.

303. A method for producing a non-human organism, comprising the steps of:

b. redifferentiating the cells to produce a non-human organism.

304. The method of claim 303, wherein the organism is a mammal.

305. The method of claim 304, wherein the mammal is a mouse.

306. A redifferentiated cell produced by the method of claim 300.

307. A non-human organism produced by the method of claim 303.

308. The non-human organism of claim 307, wherein said organism is a mammal.

309. The non-human organism of claim 308, wherein said mammal is a mouse.