MXPA00003014A - Expression of endogenous genes by non-homologous recombination of a vector construct with cellular dna - Google Patents

Abstract

The present invention relates generally to activating gene expression or causing over-expression of a gene by recombination methods in situ. The invention also relates generally to methods for expressing an endogenous gene in a cell at levels higher than those normally found in the cell. In one embodiment of the invention, expression of an endogenous gene is activated or increased following integration into the cell, by non-homologous or illegitimate recombination, of a regulatory sequence that activates expression of the gene. In another embodiment, the expression of the endogenous gene may be further increased by co-integration of one or more amplifiable markers, and selecting for increased copies of the one or more amplifiable markers located on the integrated vector. The invention also provides methods for the identification and expression of genes undiscoverable by current methods since no target sequence is necessary for integration. The invention also provides methods for isolation of nucleic acid molecules (particularly cDNA molecules) encoding transmembrane proteins, and for isolationof cells expressing such transmembrane proteins which may be heterologous transmembrane proteins. The invention also relates to isolated genes, gene products, nucleic acid molecules, and compositions comprising such genes, gene products and nucleic acid molecules, that may be used in a variety of therapeutic and diagnostic applications. Thus, by the present invention, endogenous genes, including those associated with human disease and development, may be activated and isolated without prior knowledge of the sequence, structure, function, or expression profile of the genes.

Description

EXPRESSION OF ENDOGENOUS GENES THROUGH NON-APPROVAL RECOMBINATION OF A VECTOR CONSTRUCTION WITH CELLULAR DNA BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The field of the invention is to activate the expression of a gene or cause overexpression of a gene by in situ recombination methods. The invention relates to expressing an endogenous gene in a cell at higher levels than those normally found in the cell. The expression of the gene is activated or increased after integration, by the non-homologous or illegitimate recombination of a regulatory sequence that activates the expression of the gene. The method allows the identification and expression of genes that can not be discovered by current methods, since the sequence selected for integration is not necessary. Therefore, gene products related to human diseases and development can be obtained from genes that have not been sequenced and in fact, whose existence is unknown, as well as from well-characterized genes. The methods provide gene products from said genes for therapeutic and diagnostic purposes.

BACKGROUND ART The identification and overexpression of novel genes associated with human diseases is an important step towards the development of new therapeutic drugs. Current approaches to generate cell libraries for overexpression of the protein are based on the production and cloning of cDNA. Therefore, in order to identify a new gene using this approach, the gene must be expressed in the cells that were used to generate the library. The gene must also be expressed at sufficient levels to be adequately represented in the library. This is problematic, since many genes are expressed only in very small amounts, in a rare population of cells or during short periods of development. In addition, due to the large size of some messenger RNAs, it is difficult or impossible to produce full-length cDNA molecules capable of expressing for the biologically active protein. The lack of full length cDNA molecules for minor messenger RNAs has been observed and is thought to be related to the sequences in the message and which are difficult to produce by reverse transcription or which are unstable during propagation in bacteria. As a result, even the most complete cDNA libraries express only a fraction of the entire group of possible genes. Finally, various cDNA libraries are produced in bacterial vectors. The use of these vectors to express biologically active mamalian proteins is severely limited, since most mammary proteins do not fold correctly and / or are inadequately glycosylated in bacteria. Accordingly, a method for generating a more representative library for the expression of the protein, which can facilitate the reliable expression of biologically active proteins, would be extremely valuable. Suitable methods for overexpressing proteins include cloning the gene of interest and placing it in a construct, together with a suitable promoter / enhancer, a polyadenylation signal, and a splice site, and introducing the construct into a host cell adequate An alternative approach involves the use of homologous recombination to the expression of the activated gene by selecting a strong promoter or other regulatory sequence to a previously identified gene. WO90 / 14092 describes the modification of genes in situ, in mamalia cells, coding proteins of interest. This application describes single chain oligonucleotides for site-directed modification of genes encoding proteins of interest. A marker can also be included. ver, methods for providing an oligonucleotide sequence substantially homologous to a selected site are limited. Thus, the method requires knowledge of the site that is required for activation through site-directed modification and homologous recombination. Novel genes can not be discovered by such methods. WO91 / 06667 describes methods for expressing a mamalium gene in situ. With this method, an amplifiable gene is introduced near a selected gene by homologous recombination. When the cell is then cultured in the appropriate medium, both the amplifiable gene and the selected gene are amplified and there is an increased expression of the selected gene. As in the previous case, the methods for introducing the amplifiable gene are limited to homologous recombination, and are not useful for activating novel genes whose sequence (or existence) is unknown. WO91 / 01140 describes the deactivation of endogenous genes by modifying cells by homologous recombination.

With these methods, homologous recombination is used to modify and deactivate genes and to produce cells that can be useful as donors in gene therapy. WO92 / 20808 describes methods for modifying selected genomic sites in situ. The modifications are described as being very small, for example, changing the simple bases in the DNA. The method is based on the genomic modification using a homologous DNA to select. WO92 / 19255 describes a method for improving the expression of a selected gene, which is achieved by homologous recombination, in which a DNA sequence is integrated into the genome or into the larger genomic fragment. This modified sequence can then be transferred to a secondary host for expression. An amplifiable gene can be integrated near the selected gene so that the selected region can be amplified to obtain increased expression. Homologous recombination is necessary for this selected approach. WO93 / 09222 describes methods for making proteins by activating an endogenous gene that encodes a desired product. A regulatory region is targeted by homologous recombination and the region normally associated with the gene whose expression is desired is replaced or deactivated. This deactivation or replacement causes the gene to be expressed at higher levels than normal. WO94 / 12650 describes a method for activating the expression of an endogenous gene and amplifying it in situ in a cell, whose gene is not expressed per se or is not expressed at desired levels in the cell. The cell is transfected with exogenous DNA sequences that repair, alter, suppress or replace a sequence present in the cell, or that are regulatory sequences that are not functionally linked to the endogenous gene in the cell. To do the above, DNA sequences homologous to the genomic DNA sequences are used to a site previously selected to select the endogenous gene. In addition, an amplifiable DNA encoding a selection marker can be included. By culturing the homologously recombinant cells under conditions they select for amplification, both the endogenous gene and the amplifiable marker are amplified together and the expression of the gene is increased. WO95 / 31560 discloses DNA constructs for homologous recombination. The constructs include an identification sequence, a regulatory sequence, an exon, and an unpaired splice donor site. Identification is achieved by homologous recombination of the construct with genomic sequences in the cell and allows the production of a protein in vitro or in vivo. WO96 / 29411 describes methods using an exogenous regulatory sequence, an exogenous exon, either encoding or not coding, and a splice donor site introduced at a site previously selected in the genome by homologous recombination. In this application, the introduced DNA is placed in such a way that the transcripts under control of the exogenous regulatory region include both the exogenous exon and the endogenous exons present in either thrombopoietin, Dnasa I, or interferon ß genes, which result in transcripts in which exogenous and exogenous exons are functionally linked. The novel transcription units are produced by homologous recombination. The Patent E.U.A. No. 5,272,071 describes the activation of transcription of silent genes transcriptionally in a cell, by inserting a regulatory element of DNA that can promote the expression of a gene normally expressed in that cell. The regulatory element is inserted to be functionally linked to the normally silent gene. The insertion is achieved through homologous recombination generating a DNA construct with a normally silent segment of the gene. (The selected DNA) and the DNA regulatory element used to induce the desired transcription. The Patent E.U.A. No. 5, 578,461 describes the expression of selected mamalian genes by homologous recombination. A DNA sequence is integrated into the genome or into a large genomic fragment to increase the expression of the selected gene. The modified construct can then be transferred to a secondary host cell. An amplifiable gene can be integrated adjacent to the selected gene such that the selected region is amplified to achieve increased expression. Both of these approaches (the construction of an overexpressing construct by cloning or homologous recombination in vivo) require that the gene be cloned and sequenced before it can be overexpressed. Additionally, using homologous recombination, the genomic sequence and structure can also be known. Unfortunately, many genes have not yet been identified and / or sequenced. Therefore, a method for overexpressing a gene of interest would be useful, whether it has been previously cloned or not, or that its sequence and structure have been known or not.

BRIEF DESCRIPTION OF THE INVENTION Therefore, the invention relates generally to methods for overexpressing an endogenous gene in a cell, comprising introducing a vector containing a transcription regulatory sequence within the cell, allowing the vector to integrate into the genome of the cell by non-homologous recombination, and allowing overexpression of the endogenous gene in the cell. The method does not require prior knowledge of the sequence of the endogenous gene or even of the existence of the gene. The invention also encompasses constructs of novel vectors for activating gene expression or overexpression of a gene through non-homologous recombination. The novel construction lacks homologous identification sequences. That is, it does not contain nucleotide sequences that select the host cell DNA and promote homologous recombination at the selected site, causing the overexpression of a cell gene by the introduced transcription regulatory sequence. The novel vector constructs include a vector that contains a transcriptional regulatory sequence functionally linked to an unpaired splice donor sequence and further contains one or more amplifiable labels. The novel vector constructs include constructs with a transcriptional regulatory sequence operably linked to a translation initiation codon, a secretion signal sequence and an unpaired splice donor site; constructs with a transcriptional regulatory sequence, functionally linked to a start codon of translation, an epitope tag and an unpaired splice donor site; the constructs contain a transcriptional regulatory sequence operably linked to a translation initiation codon, a signal sequence and an epitope tag, and an unpaired splice donor site; constructs containing a transcriptional regulatory sequence functionally linked to a translation initiation codon, a secretion signal sequence, an epitope tag, and a sequence specific protease site and an unpaired splice donor site. The vector construct may contain one or more selection markers for the selection of the recombinant host cell. Alternatively, selection can be effected by phenotypic selection to obtain a character provided by the activated endogenous gene product. These vectors, and indeed any of the vectors described herein, as well as the variants of the vectors that those skilled in the art will readily recognize, can be used in any of the methods described herein to form any of the compositions that They can be produced through these methods. The transcription regulatory sequence used in the vector constructions of the invention includes, but is not limited to, a promoter. In preferred embodiments, the promoter is a viral promoter. In highly preferred embodiments, the viral promoter is the cytomegalovirus immediate early promoter. In alternative embodiments, the promoter is a cellular, non-viral or inducible promoter. The transcription regulatory sequence that is used in the vector construct of the invention may also include, but is not limited to, an enhancer. In preferred embodiments, the enhancer is a viral enhancer. In highly preferred embodiments, the viral enhancer is the immediate early enhancer of cytomegalovirus. In alternative embodiments, the enhancer is a cellular non-viral enhancer. In preferred embodiments of the methods described herein, the vector construct is, or may contain, linear RNA or DNA. The cell containing the vector can be selected for the expression of the gene. The cell that overexpresses the gene can be cultured in vitro under conditions that favor the production, by the cell, of desired amounts of the gene product (also referred to indistinctly herein as "the expression product") of the endogenous gene that has been activated or whose expression has been increased. The expression product can then be isolated and purified for use, for example, in protein therapy or drug discovery. Alternatively, the cell expressing the desired gene product may be allowed to express the gene product in vivo. In certain mentioned aspects of the invention, the cell containing a vector construction of the invention integrated in its genome can be introduced in a eucahonte (such as a vertebrate, particularly a mammal and very particularly a human) under conditions that favor overexpression or activation of the gene by the cell in vivo in the eukaryote. In those related aspects of the invention, the cell can be isolated and cloned before being introduced into the eukaryote. The invention is also directed to methods for overexpressing an endogenous gene in a cell, comprising the introduction of a vector containing a transcription regulatory sequence and one or more amplifiable markers in the cell, allowing the vector to be integrated into the genome of the cell by recombination does not homologate and allowing the overexpression of the endogenous gene in the cell. The cell containing the vector can be selected for overexpression of the gene. The cell that overexpresses the gene is cultured in such a way that the amplification of the endogenous gene is obtained. The cell can then be cultured in vitro to produce desired quantities of the gene product of the amplified endogenous gene that has been activated or whose expression has been increased. The gene product can be isolated and purified. Alternatively, after amplification, the cell may be allowed to express the endogenous gene and produce desired quantities of the gene product in vivo. However, it should be understood that any vector used in the methods described herein may include one or more amplifiable markers. Accordingly, the amplification of both the vector and the DNA of interest (ie, containing the overexpressed gene) occurs in the cell, and in addition, increased expression of the endogenous gene is obtained. In accordance with the foregoing, the methods may include a step in which the endogenous gene is amplified. The invention also relates to methods for overexpressing an endogenous gene in a cell comprising introducing a vector containing a transcription regulatory sequence and an unpaired splice donor sequence in the cell, which allows the vector to integrate into the genome of the cell by non-homologous recombination, and allows overexpression of the endogenous gene in the cell. The cell containing the vector can be selected for expression of the gene. The cell that overexpresses the gene can be cultured in vitro to produce desired amounts of the gene product of the endogenous gene, whose expression has been activated or increased. The gene product can then be isolated and purified. Alternatively, the cell may be allowed to express the desired gene product in vivo. The vector construct may consist essentially of the transcription regulatory sequence. The vector construct may consist essentially of the transcriptional regulatory sequence and one or more amplifiable markers. The vector construct may consist essentially of the transcription regulatory sequence and the splice donor sequence. Any of the constructions of the vector of the invention may further include a secretion signal sequence. The secretion signal sequence is placed in the construct in such a way that it will functionally bind to the activated endogenous protein. Therefore, the secretion of the protein of interest occurs in the cell, and the purification of that protein is facilitated. In accordance with the above, the methods may include a step in which the protein expression product is secreted from the cell. The invention further relates to cells generated by any of the foregoing methods. The invention also relates to cells that contain the vector constructs, cells in which the vector constructs have been integrated into the cell genome and cells that overexpress the desired gene products from an endogenous gene, said overexpression is driven by the transcription regulatory sequence introduced. The cells can be isolated and cloned. The methods can be carried out in any cell of eukaryotic origin, such as fungal, plant or animal. In preferred embodiments, the methods of the invention can be carried out in vertebrate cells, and particularly in mammary cells which include but are not limited to cells from rats, mice, bovines, swine, sheep, goats and human cells, and very particularly in human cells. A simple cell generated by the methods described above can overexpress a single gene or more than one gene. More than one gene can be activated in a cell by integrating a simple type of construction into multiple sites in the genome. Similarly, more than one gene in a cell can be activated by integrating multiple constructs (ie, more than one type of construct) into multiple sites in the genome. Therefore, a cell can contain only one type of vector construct or different types of constructions, each of which can activate an endogenous gene. The invention also relates to methods for generating the cells that were described above by one or more of the following steps: introducing one or more of the vector constructs of the invention into a cell; allow the introduced construct (s) to be integrated into the cell genome by non-homologous recombination; allow overexpression of one or more endogenous genes in the cell; as well as isolate and clone the cell. The invention also relates to cells produced by said methods, which may be isolated cells. The invention also encompasses methods for using the cells described above to overexpress a gene, such as an endogenous cellular gene, which has been characterized (eg, sequenced), or uncharacterized, (e.g., a gene whose function is known but which it has not been cloned or sequenced), or a gene whose existence, prior to overexpression, was unknown. The cells can be used to produce desired amounts of an expression product in vitro or in vivo. If desired, this expression product can be isolated and purified, for example, by cell lysis or by isolation of the growth medium (as in the case when the vector contains a secretion signal sequence). The invention also encompasses cell libraries generated by the methods described above. A library can encompass all clones of a simple transfection experiment. The subgroup may overexpress the same gene or more than one gene, for example, a class of genes. The transfection can be carried out with a simple construction or with more than one construction. A library can also be formed by combining all of the recombinant cells from two or more transfection experiments, combining one or more subsets of cells from a single transfection experiment or by combining cell subgroups from transfection experiments performed separately. The resulting library can express the same gene, or more than one gene, for example, a class of genes. Again, in each of the individual transfections, a single construction or more than one construction can be used.

The libraries can be formed from the same cell type or from different cell types. The invention also relates to methods for forming libraries by selecting various subgroups of cells from the same or different transfection experts. The invention also relates to methods for using the cells or cell libraries described above, for overexpressing or activating endogenous genes, or for obtaining the gene expression products of said overexpressed or activated genes. According to this aspect of the invention, the cell or library can be selected for the expression of the gene, just as the cells expressing the desired gene product can be selected. Then the cell can be used to isolate or purify the gene product for subsequent use. Expression in the cell can occur by culturing the cell in vitro, under conditions that favor the production of the endogenous gene expression product by the cell, or by allowing the cell to express the gene in vivo. In preferred embodts of the invention, the methods include a method wherein the expression product is isolated or purified. In highly preferred embodts, cells expressing the endogenous gene product are cultured under conditions that favor the production of sufficient quantities of the gene product for commercial purposes, and especially for diagnostic uses, drug discovery and therapeutic uses. Any of the methods may further comprise the introduction of double-strand breaks in the genomic DNA in the cell before or simultaneously with the integration of the vector. Other preferred embodts of the present invention will be apparent to those skilled in the art in light of the following drawings and description of the invention, as well as the claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1.- Schematic diagram of the activation events of genes described in the present. The activation construct is transfected into the cells and allowed to integrate into the chromosomes of the host cell to DNA breaks. If the break upstream of a gene of interest occurs (e.g., Epo) and the proper activation construct is integrated into the disruption such that its regulatory sequence is functionally linked to the gene of interest, then activation of the gene will be presented. Transcription and splicing produce a chic RNA molecule that contains exonic sequences from the activation construct and from the endogenous gene. Subsequent translation will result in the production of the protein of interest. After isolation of the recombinant cell, gene expression can be further increased by amplification of the gene.

Figure 2.- Schematic diagram of activation constructions not translated. The arrows show the promoter sequences. Exon sequences are shown as open boxes and the splice donor sequence is indicated by S / D (D / E). The construction numbers corresponding to the following description are shown on the left side. The selection and amplification markers are not displayed. Figure 3.- Schematic diagram of translated activation constructions. The arrows show the promoter sequences. The exon sequences are shown as open boxes and the splice donor sequence is indicated by S / D (D / E). Translated sequences of signal peptide, epitope tag, and protease cut sequences are shown in the legend below the constructs. The building numbers corresponding to the following description are shown on the left side. The selection and amplification markers are not displayed. Figure 4. Schematic diagram of an activation construct that can activate endogenous genes. Figures 5A-5D. Nucleotide sequence of pRIG8R1-CD2 (SEQ ID NO: 7). Figures 6A-6C. Nucleotide sequence of pRIG8R2-CD2 (SEQ ID NO: 8). Figures 7A-7C. Nucleotide sequence of pRIG8R3-CD2 (SEQ ID NO: 9).

DETAILED DESCRIPTION OF THE INVENTION There are great advantages of genetic activation through non-homologous recombination compared with other genetic activation procedures. Unlike previous methods of protein overexpression, the methods described herein do not require that the gene of interest be cloned (isolated from the cell). Nor do they require any knowledge of the DNA sequence or structure of the gene to be overexpressed (ie, the sequence of ORF (open reading frame), introns, exons, or regulatory elements upstream and downstream) or knowledge of gene expression patterns (ie, specific tissue character, developmental regulation, etc.). Moreover, the methods do not require any knowledge regarding the genomic organization of the gene of interest (ie, the structure of the intron and exon). The methods of the present invention therefore involve vector constructs that do not contain nucleotide sequences selected for homologous recombination. A selected sequence allows the homologous recombination of vector DNA with cellular DNA at a predetermined site in the cellular DNA, the site has homology to the sequences in the vector, homologous recombination at the predetermined site results in the introduction of the regulatory sequence of transcription in the genome and the subsequent activation of endogenous gene. The method of the present invention does not include integration of the vector into predetermined sites. In contrast, the methods of the present invention involve the integration of the vector constructs of the invention into cellular DNA (eg, the cell genome) by non-homologous or "illegitimate" recombination. The vectors described herein do not contain selected sequences. A selected sequence is a sequence in the vector that has homology to a sequence or sequences within the gene to be activated or upstream of the gene to be activated, the upstream region that is above, and including the first functional splice acceptor site in the same coding chain of the gene of interest, and whereby the homology of the transcription regulatory sequence that activates the gene of interest is integrated into the genome of the cell that contains the gene that is to be activated. In the case of an enhancer integration vector that activates an endogenous gene, the vector does not contain homology to any sequence in the genome upstream or downstream of the gene of interest (or within the gene of interest) for such a distance far as the enhancing function is operational. Therefore, the present methods are capable of identifying new genes that have been omitted or that can be omitted using conventional and currently available cloning techniques. By using the constructs and methodology described herein, one can easily identify unknown and / or uncharacterized genes that have been overexpressed to produce proteins. The proteins have uses such as, among others, therapeutic and diagnostic in humans and as targets for the discovery of drugs. The described methods are also capable of producing the overexpression of genes known and / or characterized for the production of proteins in vitro or in vivo. A "known" gene refers to the level of characterization of a gene. The invention allows the expression of genes that have been characterized, as well as the expression of genes that have not been characterized. It is possible to have different levels of characterization. These include detailed characterization, such as cloning, DNA, RNA, and / or protein sequencing, and in relation to the regulation and function of the gene to the cloned sequence (e.g., recognition of the promoter and enhancer sequences)., functions of open reading frames, introns and the like). The characterization may be less detailed, such as having mapped a gene and its related function, or having a partial amino acid or nucleotide sequence, or having purified a protein and investigated a function. The characterization may be minimal, as in the case when a nucleotide or amino acid sequence is known or a protein has been isolated but the function is unknown. Alternatively, a function may be known but the associated protein or nucleotide sequence is not known or is known, but has not been correlated with function. Finally, there may be no characterization, when the existence and function of the gene are not known. The invention allows the expression of any gene in any of these or other specific characterization grades. Many different proteins can be activated or overexpressed (also referred to herein interchangeably as "gene products" or "expression products") by a simple activation construct and in a single group of transfections. Therefore, a single cell or different cells in a group of transfectants (library) can overexpress more than one protein after transfection with the same constructs or with different constructions. The previous activation methods require a unique construction to be generated for each gene that is to be activated. In addition, various different integration sites adjacent to a single gene can be generated, and can be tested simultaneously using a single construct. This allows the rapid determination of the optimal genomic position of the activation construct for the expression of the protein. Using the above methods, the 5 'end of the gene of interest had to be characterized extensively with respect to sequence and structure. In order for each activation construct to occur, an appropriate selected sequence must be isolated. Generally, this must be an isogenic sequence isolated from the same person or laboratory animal strain according to the type of cells that will be activated. In some cases, this DNA can be 50 kb or more of the gene of interest. Therefore, the production of each identification construct required a difficult amount of cloning and sequencing of the endogenous gene. However, since the sequence and structure information for the methods of the present invention is not required, unknown genes and genes with uncharacterized upstream regions can be activated. This is possible using in situ gene activation using non-homologous recombination of exogenous DNA sequences with cellular DNA. The methods and compositions (eg, vector constructs) that are required to achieve such in situ gene activation using non-homologous recombination are contemplated by the present invention. DNA molecules can recombine to redistribute their genetic content by several different and distinct mechanisms, including homologous recombination, site-specific recombination, and non-homologous / illegitimate recombination. Homologous recombination involves recombination between DNA spaces that are very similar in sequence. It has been shown that homologous recombination involves matching between the homologous sequences along their length prior to the redistribution of the genetic material. The exact site of crosslinking can be any point in the homologous segments. The efficiency of recombination is proportional to the length of homologous selected sequences (Hope, Development 113: 399 (1991J; Reddy et al., J. Virol. 65A 507 (1991)), the degree of sequence identity between the two recombinant sequences (von Melchner et al., Genes Dev. 6: 919 (1992)), and the ratio of DNA homologous to non homologous present in the construction (Letson, Genetics 777: 759 (1987)). On the other hand, site-specific recombination involves the exchange of genetic material at a predetermined site, designated by specific DNA sequences. In this reaction, a recombinase protein binds to the recombination signal sequences, generates a chain excision, and facilitates the exchange of DNA strands. Cre / Lox recombination is an example of site-specific recombination. Non-homologous / illegitimate recombination such as that which is conveniently used by means of the methods of the present invention, involves the binding (exchange or redistribution) of genetic material that does not share significant sequence homology and does not occur in the sequences of site-specific recombination. Examples of non-homologous recombination include the integration of exogenous DNA into chromosomes at non-homologous sites, translocations and chromosomal deletions, DNA end binding, repair of double-stranded chromosomal end breaks, bridging-breaking fusion, and concatemerization of transfected sequences . In most cases, it is considered that non-homologous recombination occurs through the binding of "free DNA ends". Free ends are DNA molecules that contain an end that can be attached to a second DNA and either directly, or after repair or processing. The DNA terminus may consist of a 5 'pendant end, 3' pendant, or shaved end. As used herein, retroviral insertion and other transposition reactions are vaguely considered forms of non-homologous recombination. These reactions do not involve the use of homology between the recombinant molecules. Moreover, unlike site-specific recombination, these types of recombination reactions do not occur between discrete sites. Instead, a specific protein / DNA complex is required in only one of the recombination elements (ie, the retrovirus or transposon), with the second DNA element (ie, the cell genome) that is generally not It is relatively specific. As a result, these "vectors" are not integrated into the cell genome in a selected form, and therefore can be used to provide the activation construct according to the present invention. Useful vector constructs for the methods described herein may ideally contain a transcriptional regulatory sequence that undergoes non-homologous recombination with genomic sequences in a cell to overexpress an endogenous gene in that cell. The vector constructs of the invention also lack homologous identification sequences. That is, they do not contain DNA sequences that select the DNA of the host cell and promote homologous recombination at the selected site. Therefore, the integration of the vector constructs of the present invention into the cell genome occurs by means of non-homologous recombination and can lead to the overexpression of a cellular gene by the introduced transcription regulatory sequence that is contained in the construction of integrated vector. The invention generally relates to methods for overexpressing an endogenous gene in a cell, comprising the introduction of a vector containing a transcription regulatory sequence within the cell, allowing the vector to be integrated into the cell genome by non-recombination. homologous and thus allowing overexpression of the endogenous gene in the cell. The method does not require prior knowledge of the sequence of the endogenous gene or even of the existence of the gene. However, when you know the sequence of the gene that is going to be activated, the constructs can be genetically manipulated to contain the appropriate configuration of the vector elements (eg, position of the start codon, addition of codons present in the first exon of the endogenous gene, and the appropriate reading frame) to achieve maximum overexpression and / or the appropriate protein sequence. In certain embodiments of the invention, the cell containing the vector can be selected for the expression of the gene. The cell that overexpresses the gene can be cultured in vitro under conditions that favor the production, of the cell, of desired amounts of the gene product of the endogenous gene that has been activated or whose expression has been increased. If desired, the gene product can be isolated or purified for use, for example in protein therapy or drug discovery. Alternatively, the cell expressing the desired gene product may be allowed to express the gene product in vivo. The vector construct may consist essentially of the transcription regulatory sequence. Alternatively, the vector construct may consist essentially of the transcriptional regulatory sequence and one or more amplifiable markers. Accordingly, the invention also relates to methods for overexpressing an endogenous gene in a cell, comprising the introduction of a vector containing a transcriptional regulatory sequence and an amplifiable marker within the cell, allowing the vector to be integrated into the cell. cell genome by non-homologous recombination, and allowing overexpression of the endogenous gene in the cell. The cell containing the vector is selected for overexpression of the gene. The cell that overexpresses the gene is cultured in such a way that the amplification of the endogenous gene is obtained. The cell can then be cultured in vitro to produce the desired amounts of the gene product of the amplified endogenous gene that has been activated or whose expression has been increased. The gene product can then be isolated and purified. Alternatively, after amplification, the cell can be allowed to express the endogenous gene and produce desired amounts of the gene product in vivo. The vector construct may consist essentially of the transcription regulatory sequence and the splice donor sequence. Therefore, the invention also encompasses methods for overexpressing an endogenous gene in a cell comprising the introduction of a vector containing a transcription regulatory sequence and an unpaired splice donor sequence in the cell, allowing the vector to integrate into the genome of the cell by non-homologous recombination, and allowing the overexpression of the endogenous gene in the cell. The cell containing the vector is selected for the expression of the gene. The cell that overexpresses the gene can be cultured in vitro to produce desirable quantities of the gene product of the endogenous gene whose expression has been activated or increased. The gene product can then be isolated and purified. Alternatively, the cell may be allowed to express the desired gene product in vivo. The vector construct can consist essentially of a transcriptional regulatory sequence functionally linked to an unpaired splice donor sequence and further containing an amplifiable label. Other activation vectors include constructs with a transcriptional regulatory sequence and an exonic sequence containing a start codon; a transcription regulatory sequence and an exonic sequence containing a start codon of translation and a secretion signal sequence; constructs with a transcriptional regulatory sequence and an exonic sequence containing a start codon of translation and an epitope tag; constructs containing a transcriptional regulatory sequence and an exonic sequence containing a start codon of translation, a signal sequence and an epitope tag; constructs containing a transcriptional regulatory sequence and an exonic sequence with a translation start codon, a secretion signal sequence, an epitope tag and a specific protease sequence site. In each of the above constructions, the exon in the construction is located immediately upstream of the unpaired splice donor site. The constructs may also contain a regulatory sequence, a selectable marker that lacks a poly A signal, an internal ribosome entry site ("res"), and an unpaired splice donor site (FIG. 4). Optionally, a start codon, a secretion signal sequence, an epitope tag, and / or a protease cleavage site may be included between the internal ribosome entry sites (ires) and the unpaired splice donor sequence. When this construct is integrated upstream of a gene, the selectable marker will be expressed efficiently, since the poly A site will be provided by the endogenous gene. In addition, the downstream gene will also be expressed because the internal ribosome entry sites (ires) will allow translation to the protein to start in the downstream open reading frame (ie, the endogenous gene). Therefore, the message produced by this activation construct will be polycistronic. The advantage of this construction is that integration events that do not occur near the genes and in the proper orientation will not produce a drug-resistant colony. The reason for this is that without a poly A end (supplied by the endogenous gene), the neomycin resistance gene will not be expressed efficiently. By reducing the number of non-productive integration events, the complexity of the library can be reduced without affecting its coverage (the number of activated genes) and this will facilitate the selection procedure. In another embodiment of this construct, cre-lox recombination sequences may be included between the regulatory sequence and the neo start codon and between the internal ribosome entry sites (ires) and the unpaired splice donor site (among the internal ribosome entry sites (ires) and the start codon if present). After isolation of cells that have activated the gene of interest, the neo gene and the internal ribosome entry sites (ires) can be removed by transfecting the cells with a plasmid encoding recombinase ere. This would eliminate the production of the polycistronic message and allow the endogenous gene to be expressed directly from the regulatory sequence in the integrated activation construct. The use of ere recombination has been described to facilitate the deletion of genetic elements from mamalian chromosomes in (Gu al, Science 265: 103 (1994), Sauer, Meth, Enzymology 225: 890-900 (1993)). Therefore, constructs useful in the methods described herein include, but are not limited to, the following: (See also Figures 1-4): 1) Construction with a regulatory sequence and an exon lacking a codon of the start of the translation. 2) Construction with a regulatory sequence and an exon that lacks a start codon of translation followed by a splice donor site. 3) Construction with a regulatory sequence and an exon containing a translation initiation codon in reading frame 1 (in relation to the splice donor site), followed by an unpaired splice donor site. 4) Construction with a regulatory sequence and an exon containing a translation initiation codon in reading frame 2 (in relation to the splice donor site), followed by an unpaired splice donor site.

) Construction with a regulatory sequence and an exon containing a translation initiation codon in reading frame 3 (in relation to the splice donor site), followed by an unpaired splice donor site. 6) construction with a regulatory sequence and an exon containing an initiation codon of translation and a signal secretion sequence in reading frame 1 (relative to the splice donor) site followed by an unpaired site donor splice. 7) Construction with a regulatory sequence and an exon containing an initiation codon of translation and a signal secretion sequence in reading frame 2 (relative to the splice donor site), followed by a site unpaired of donor splice. 8) Construction with a regulatory sequence and an exon containing an initiation codon of translation and a signal secretion sequence in reading frame 3 (relative to the splice donor site), followed by an unpaired site donor splice. 9) Construction with a regulatory sequence and an exon containing (from 5 'to 3') a start codon of translation and an epitope tag in reading frame 1 (relative to the splice donor site) followed by an unpaired site of a splice donor. 10) Construction with a regulatory sequence and an exon containing (from 5 'to 3') a start codon of translation and an epitope tag in reading frame 2 (relative to the splice donor site) followed by an unpaired site of a splice donor. 11) Construction with a regulatory sequence and an exon containing (from 5 'to 3') a start codon of translation and an epitope tag in reading frame 3 (relative to the splice donor site) followed by an unpaired site of a splice donor. 12) Construction with a regulatory sequence and an exon containing (from 5 'to 3'), a start codon of the translation, a secretion signal sequence, and an epitope tag in reading frame 1 (relative to the splice donor site), followed by an unpaired splice donor site. 13) Construction with a regulatory sequence and an exon containing (from 5 'to 3') a translation initiation codon, a secretion signal sequence, and an epitope tag in reading frame 2 (relative to the splice donor site), followed by an unpaired splice donor site. 14) Construction with a regulatory sequence and an exon containing (from 5 'to 3'), a translation initiation codon, a secretion signal sequence, and an epitope tag in reading frame 3 (relative to the splice donor site), followed by an unpaired splice donor site. 15) Construction with a regulatory sequence and an exon containing (from 5 'to 3'), a start codon of the translation, a secretion signal sequence, an epitope tag, and a sequence specific protease site in the reading frame 1 (in relation to the splice donor site), followed by an unpaired splice donor site. 16) Construction with a regulatory sequence and an exon containing (from 5 'to 3'), a start codon of the translation, a secretion signal sequence, an epitope tag, and a sequence specific protease site in the reading frame 2 (in relation to the splice donor site), followed by an unpaired splice donor site. 17) Construction with a regulatory sequence and an exon containing (from 5 'to 3'), a start codon of the translation, a secretion signal sequence, an epitope tag, and a sequence specific protease site in the reading frame 3 (in relation to the splice donor site), followed by an unpaired splice donor site. 18) Construction with a regulatory sequence linked to a selectable marker, followed by an internal ribosome entry site, and an unpaired splice donor site. 19) Construction 18 in which a recombination signal cre / lox enter locates) the regulatory sequence and the open reading frame of the selectable marker and b) between internal sites ribosome entry (IRES) and the site unpaired of splice donor 20) Construction with a regulatory sequence operably linked to an exon containing green fluorescent protein lacking a stop codon, followed by an unpaired splice donor site. However, it should be understood, that any vector used in the methods described herein may include one or more (ie, one, two, three, four, five, or more and most preferably one or two) amplifiable markers. In accordance with the foregoing, the methods may include a step in which the endogenous gene is amplified. The placement of one or more amplifiable markers in the activation construct results in the juxtaposition of the gene of interest and one or more amplifiable markers in the activated cell. Once the activated cell has been isolated, the expression can be further increased by selecting those cells that contain an increased copy number of the locus that contains both the gene of interest and the activation construct. This can be achieved by selection methods known in the art, for example by culturing cells in selective culture media containing one or more selection agents that are specific for one or more amplifiable markers contained in the genetic construct or vector. After activation of an endogenous gene by non-homologous integration of any of the vectors described above, the expression of the endogenous gene can also be increased by selecting the increased copies of the localized amplifiable marker (s) in the integrated vector. Although such an approach can be achieved using an amplifiable marker in the integrated vector, in an alternative embodiment of the invention, said methods are provided wherein two or more (i.e., two, three, four, five, or more, and most preferably two) ) amplifiable markers can be included in the vector to facilitate the most efficient selection of cells that have amplified the vector and the flanking gene of interest. This approach is particularly useful in cells that have a functional endogenous copy of one or more of the amplifiable marker (s) that are contained in the vector, since the selection procedure can result in the isolation of cells that have incorrectly amplified the endogenous amplifiable marker (s) in place of the amplifiable marker (s) encoded by vector. This approach is also useful for screening against cells that develop resistance to the selective agent by mechanisms that do not involve amplification of the gene. The approach using two or more amplifiable markers has great advantage in these situations due to the probability of a cell to develop resistance to two or more selective agents (resistance encoded by two or more amplifiable markers) without amplifying the integrated vector and the flanking of interest that is significantly less than the probability of the cell to develop resistance to any individual selective agent. Therefore, when selecting for two or more vector-encoded amplifiable markers, either simultaneously or sequentially, a higher percentage of cells that were finally isolated will contain the amplified vector and the gene of interest. Therefore, in another embodiment, the vectors of the invention may contain two or more (ie, two, three, four, five, or more, and most preferably two) amplifiable labels. This approach allows a more efficient amplification of the sequences of the vector and of the adjacent gene of interest after the activation of expression. Examples of amplifiable labels that can be used to construct the present vectors include, but are not limited to, dihydrofolate reductase, adenosine deaminase, aspartate transcarbamylase, dihydro-orotase, and carbamyl phosphate synthase. It is also understood that any of the constructions described herein may contain a replication eukaryotic viral origin, either in place of, or in conjunction with, an amplifiable marker. The presence of the viral origin of replication allows the integrated vector and the adjacent endogenous gene to be isolated as an episome and / or amplified to a larger number of copies upon introduction of the appropriate viral replication protein. Examples of useful viral sources are included, more are not limited to, ori SV40 and oñ P EBV. The invention also encompasses embodiments in which the constructions described herein consist essentially of the components that are specifically described for these constructions. It is also understood that the above constructions are examples of constructions useful in the methods described herein, but that the invention encompasses functional equivalents of said constructions. It is understood that the term "vector" generally refers to the vehicle by which the nucleotide sequence is introduced into the cell. The intention is not to limit it to a specific sequence. The vector may itself be the nucleotide sequence that activates the endogenous gene or may contain the sequence that activates the endogenous gene. Therefore, the vector may simply be a linear or circular polynucleotide containing essentially only those sequences necessary for activation, or these sequences could be in a major polynucleotide or other construct such as a viral DNA or RNA genome or complete virion, or another biological construct that is used to introduce the critical nucleotide sequences into a cell. The vector may contain DNA sequences that exist in nature or that have been generated by manipulation of genetic engineering or synthetic procedures. When non-homologous integration is carried out within the genome of a cell, the construct can activate the expression of an endogenous gene. Expression of the endogenous gene may result in the production of long-lived protein or the production of a biologically truncated active form of the endogenous protein, depending on the site of integration (e.g., upstream region against intron 2). The activated gene may be a known gene (for example, cloned or previously characterized) or an unknown gene (not previously cloned or characterized). The function of the gene can be known or unknown. Examples of proteins with known activities include, but are not limited to, cytokines, growth factors, neurotransmitters, enzymes, structural proteins, cell surface receptors, intracellular receptors, hormones, antibodies and transcription factors. Specific examples of known proteins that can be produced by this method include, but are not limited to, erythropoietin, insulin, growth hormone, glucocerebrosidase, tissue plasminogen activator, granulocyte colony stimulation factor (G-CSF), stimulation of granulocyte / macrophage colonies (GM / CSF), interferon a, interferon beta, interferon beta, interleukin-2, interleukin-3, interleukin-4, interleukin-6, interleukin-8, interleukin-10, interleukin-11, interleukin -12, interleukin-13, interleukin-14, TGF-β, blood coagulation factor V, blood coagulation factor VII, blood coagulation factor VIII, blood coagulation factor IX, blood coagulation factor X, TSH-β, bone growth factor-2, bone growth factor-7, tumor necrosis factor, alpha-1 antitrypsin, anti-thrombin III, leukemia inhibitory factor, glucagon, protein C, protein kinase C, factor ulation of macrophage colonies (M-CSF), stem cell factor, follicle stimulating ß hormone, urokinase, nerve growth factors, growth factors in the form of insulin, insulinotropin, parathyroid hormone, lactoferrin, complement inhibitors, factor of growth derived from platelets, keratinocyte growth factor, hepatocyte growth factor, endothelial cell growth factor, neurotropin-3, thrombopoietin, chorionic gonadotropin, thrombomodulin, alpha glucosidase, epidermal growth factor, and fibroblast growth factor. The invention also allows the activation of a variety of genes that express transmembrane proteins, and the production and isolation of said proteins, including but not limited to, cell surface receptors for growth factors, hormones, neurotransmitters and cytokines such as those described above, transmembrane ion channels, cholesterol receptors, lipoprotein receptors, (including LDL and HDL) and other portions of lipids, integrins and other extracellular matrix receptors, cytoskeletal anchoring proteins, immunoglobulin receptors, CD antigens (including the CD2, CD3, CD4, CD8, and CD34 antigens) and other structural proteins and functional cell surface transmembranes that are known in the art. As will be appreciated by those skilled in the art, other cellular proteins and receptors that are known in the art can also be produced by the methods of the invention. One of the advantages of the method described herein is that virtually any gene can be activated. However, since the genes have different genomic structures, including different limits and extron / exon positions of the start codons, a variety of activation constructs are provided to activate the maximum number of different genes within a population of cells. These constructs can be transfected separately in the cells to produce libraries. Each library contains cells with a unique group of activated genes. Some genes will be activated by various different activation constructs. In addition, portions of a gene can be activated to produce truncated, biologically active proteins. Truncated proteins can be produced, for example, by integrating an activation construct into introns or exons in the middle part of an endogenous gene instead of upstream of the second exon. The use of different constructions also allows the activated gene to be modified to contain new sequences. For example, a secretion signal sequence may be included in the activation construct to facilitate secretion of the activated gene. In some cases, depending on the intron / exon structure or the gene of interest, the secretion signal sequence can replace the entire signal sequence or part of it of the endogenous gene. In other cases, the signal sequence will allow a protein that is normally located intracellularly to be secreted. The regulatory sequence in the vector can be a constitutive promoter. Alternatively, the promoter may be inducible. The use of inducible promoters will allow the activated protein to be produced at low base levels by the cell during the culture and expansion routine. The cells can then be induced to produce large amounts of the desired proteins, for example during manufacture or selection.

Examples of promoter promoters may include, but are not limited to, the inducible tetracycline promoter and the metallothionein promoter. In preferred embodiments of the invention, the regulatory sequence in the vector can be a specific tissue promoter or an enhancer. The regulatory sequence in the vector can be isolated from cellular or viral genomes. Examples of cellular regulatory sequences include, but are not limited to, regulatory elements of the actin gene, metallothionein I gene, immunoglobulin genes, casein I gene, serum albumin gene, collagen gene, globin genes, gene of laminin, spectrin gene, ancynin gene, sodium-potassium ATPase gene and tubulin gene. Examples of viral regulatory sequences include, but are not limited to, regulatory elements of the immediate gene of Cytomegalovirus (CMV), late adenovirus genes, SV40 genes, retroviral LTR, and Herpesvirus genes. Typically, the regulatory sequences contain binding sites for transcription factors such as, NF-kB, SP-1, TATA-bound protein, AP-1 and CAAT-bound protein. Functionally, the regulatory sequence is defined by its ability to promote, increase or otherwise alter the transcription of an endogenous gene. In preferred embodiments, the regulatory sequence is a viral promoter. In highly preferred embodiments, the promoter is the promoter of the immediate early CMV gene (cytomegalovirus). In alternative embodiments, the regulatory element is a cellular, non-viral promoter. In preferred embodiments, the regulator element contains an enhancer. In highly preferred embodiments, the enhancer is the enhancer of the cytomegalovirus immediate early gene. In preferred embodiments, the enhancer is a cellular, non-viral enhancer. The transcription regulatory sequence may be composed of scaffold binding regions of matrix binding sites, negative regulatory elements, and transcription factor binding sites. Regulatory sequences may also include regions of locus control. The invention encompasses the use of regulatory retroviral transcription sequences, for example, prolonged terminal repeats.

However, where the latter are used, they are not necessarily linked to any retrovirus sequence that materially affects the function of the transcription regulatory sequence as a promoter or transcription enhancer of the endogenous gene to be activated (i.e., the cell with which the transcription regulatory sequence is recombined to activate it). The construct may contain a regulatory sequence that is not operably linked to exonic sequences in the vector. For example, when the regulatory element is an enhancer, it can be integrated near an endogenous gene (e.g., upstream, downstream or in an intron) and stimulate the expression of the gene from its endogenous promoter. By means of this activation mechanism, the exonic sequences of the vector are absent in the transcription of the activated gene. Alternatively, the regulatory element can be operably linked to an exon. The exon may be a sequence that occurs naturally or may be a sequence that does not occur naturally (for example, produced synthetically). To activate endogenous genes lacking an initiation codon in their first exon (eg, hormone that stimulates follicle-β), a start codon is preferably omitted for the exon in the vector. To activate endogenous genes containing a start codon in the first exon (e.g., erythropoietin and growth hormone), the exon in the vector preferably contains a start codon, generally ATG and preferably an efficient translation start site ( Kozak, J. Mol Biol. 196: 947 (1987)). The exon may contain additional codons after the start codon. These codons can be derived from a gene that occurs naturally or it may not be naturally occurring (for example, synthetic). The codons may be the same codons as those present in the first exon of the endogenous gene to be activated. Alternatively, the codons may be different than the codons present in the first exon of the endogenous gene. For example, the codons can unify an epitope tag, secretion signal sequence, transmembrane domain, selectable marker, or selectable marker. Optionally, an unpaired splice donor site may be present immediately 3 'of the exonic sequence. When the structure of the gene to be activated is known, the splice donor site must be placed adjacent to the vector exon at a position such that the codons in the vector will be in frame with the codons of the second exon of the endogenous gene after the codon. splice. When the structure of the endogenous gene to be activated is not known, separate constructions are used, each containing a different reading frame. Operably linked is defined as a configuration that allows transcription through the designated sequence (s). For example, a regulatory sequence that is operably linked to an exonic sequence indicates that the exonic sequence is transcribed. When a start codon is present in the vector, operably linked means that the open reading frame of the vector exon is in a frame with the open reading frame of the endogenous gene. After non-homologous integration, the regulatory sequence (eg, a promoter) in the vector becomes operably linked to an endogenous gene and facilitates the initiation or initiation of transcription, to a site generally referred to as a CAP site. The procedures of transcription through the exonic elements in the vector (and, if present, through the start codon, the open reading frame, and / or the unpaired splice donor site), and through the gene endogenous. The primary transcript produced by this operable linkage is spliced to generate a chimeric transcript that contains exonic sequences of both the vector and the endogenous gene. This transcription can produce the endogenous protein when it is translated. An exon or "exon sequence" is defined as any transcribed sequence that is present in the mature RNA molecule. The exon in the vector may contain untranslated sequences, e.g., a 5 'untranslated region. Alternatively, in conjunction with the untranslated sequences, the exon may contain coding sequences such as a start codon and an open reading frame. The open reading frame can encode amino acid sequences that occur naturally and amino acid sequences that occur unnaturally (eg, synthetic codons). The open reading frame may also encode a secretory signal sequence, epitope tag, exon, selectable marker, selectable marker, or nucleotides that function to allow the open reading frame to be conserved when spliced to an endogenous gene. The splicing of primary transcripts, the procedure by which introns are removed, is directed by a splice donor site and a splice acceptor site, located at the 5 'and 3' ends of introns respectively. The consensus sequence for the splice donor sites is (A / C) AG GURAGU (where R represents a purine nucleotide) with nucleotides at positions 1-3 located in the exon and GURAGU nucleotides located in the intron. An unpaired splice donor site is defined herein as a splice donor site present in the activation construct without a downstream splice acceptor site. When the vector is integrated by non-homologous recombination into a host cell genome, the unpaired site of splice donor becomes paired with a splice acceptor site of an endogenous gene. The vector splice donor site, together with the splicing acceptor site of the endogenous gene, will then lead to cleavage of all sequences between the vector splice donor site and the endogenous splice acceptor site. The excision of these intervening sequences removes the sequences that interfere with the translation of the endogenous protein. The terms "upstream" and "downstream", as used herein, mean toward the direction (toward the end) 5 'or toward the direction (toward the end) 3', respectively, in relation to the encoded chain. The term "upstream region" of a gene is defined as the 5 'nucleic acid sequence of its second exon (relative to the coding strand) up to and including the last exon of the first adjacent gene having the same coding strand . Functionally, the upstream region is any 5 'site of the second exon of an endogenous gene that can allow a non-homologously integrated vector to be operably linked to the endogenous gene. The vector construct may contain a selectable marker to facilitate identification and isolation of cells that contain an integrated activation construct in a non-homologous manner. Examples of selectable markers include genes coding for resistance to neomycin (neo), hypoxanthine phosphoribosyl transferase (HPRT) puromycin (pac), dihydro-orotase glutamine synthetase (GS), histidine D (his D), carbamyl phosphate synthetase (CAD), dihydrofolate reductase (DHFR), drug resistance 1 (mdr 1), aspartate transcarbamylase, xanthine-guanine phosphoribosyl transferase (gpt) and adenosine deaminase (ada). Alternatively, the vector may contain a selection marker, instead of, or in addition to, the selectable marker. The selection marker allows the cells containing the vector to be isolated without placing them under drug pressures or other selective pressures. Examples of selection markers include genes encoding cell surface proteins, fluorescent proteins, and enzymes. The vector containing cells can be isolated, for example by FACS using fluorescently labeled antibodies to the cell surface protein or to substrates that can be converted to fluorescent products by a vector-encoded enzyme. Alternatively, selection can be effected by phenotypic selection to obtain a character provided by the endogenous gene product. Therefore, the activation construct may lack a selectable marker different from the "marker" provided by the endogenous gene itself. In this embodiment, the activated cells can be selected based on a phenotype conferred by the activated gene. Examples of selectable phenotypes include cell proliferation, growth factor-independent growth, colony formation, cell differentiation, (eg, differentiation within a neuronal cell, muscle cell, epithelial cell, etc.), growth independent of anchoring, activation of cellular factors, (e.g., kinases, transcription factors, nucleases, etc.), expression of cell surface / protein receptors, gain or loss of cell-to-cell adhesion, migration, and cellular activation (e.g. resting T cells against activated T cells). A selectable marker of the construct can also be omitted when the transfected cells are selected for gene activation products without selecting the stable integrating elements. This is particularly useful when the efficiency of stable integration is very high. The vector may contain one or more (i.e., one, two, three, four, five, or more, and most preferably one or two) amplifiable markers to allow the selection of cells containing increased copies of the integrated vector and the endogenous gene activated adjacent. Examples of amplifiable markers include, but are not limited to, dihydrofolate reductase (DHFR), adenosine deaminase (ada), dihydro-orotase, glutamine synthetase (GS), and carbamyl phosphate synthase (CAD). The vector may contain replication useful eukaryotic viral origins for the amplification of the gene. These origins may be present in place of, or in conjunction with, an amplifiable marker. The vector may also contain genetic elements useful for the propagation of the construction in microorganisms. Examples of useful genetic elements include microbial origins of replication and markers of antibiotic resistance. These vectors, and any of the vectors described herein, and obvious variants recognized by those skilled in the art, can be used in any of the methods described above to form any of the compositions that can be produced by those methods. The non-homologous integration of the construct into the genome of a cell causes the operable linkage between the regulatory elements of the vector and the exons from an endogenous gene. In preferred embodiments, the insertion of the vector regulatory sequences is used to upregulate the expression of the endogenous gene. Overregulation of gene expression includes converting a silent gene transcriptionally to a transcriptionally active gene. It also includes the increase of gene expression for genes that are already transcriptionally active, but that produce protein at levels lower than desired. In other embodiments, the expression of the endogenous gene may be affected in other ways such as the down-regulation of expression, the generation of an inducible phenotype or the change of the specific character of the expression tissue. According to the invention, in vitro methods of producing an expression product of a gene can comprise, for example, a) introducing a vector of the invention into a cell; b) allow the vector to integrate into the cell genome by non-homologous recombination; c) allowing overexpression of an endogenous gene in the cell by overregulating the gene by the transcriptional regulatory sequence contained in the vector; d) selecting the cell for overexpression of the endogenous gene; and e) culturing the cell under conditions that favor the production of the expression product of the endogenous gene by the cell. Said in vitro methods of the invention may further comprise isolating the expression product to produce an expression product of the isolated gene. In such methods, any method known in the art of protein isolation can be conveniently used, including but not limited to, chromatography (e.g., HPLC, FPLC, LC, ion exchange, affinity, size exclusion, and the like). ), precipitation (eg, precipitation of ammonium sulfate, immunoprecipitation and the like), electrophoresis and other methods of isolation and purification of proteins that will be known to those skilled in the art. Analogously, in vivo methods of producing a gene expression product may comprise, for example, a) introducing a vector of the invention into a cell; b) allow the vector to integrate into the cell genome by non-homologous recombination; c) allowing overexpression of an endogenous gene in the cell by overregulating the gene by the transcriptional regulatory sequence contained in the vector; d) selecting the cell for overexpression of the endogenous gene; and e) introducing the isolated and cloned cell into a eukaryote under conditions that favor overexpression of the endogenous gene by the cell in vivo and the eukaryote. According to this aspect of the invention, any eukaryote can be conveniently used, including plant and animal fungi (particularly yeast), most preferably animals, most preferably still vertebrates, and still most preferably mammals, particularly humans. In certain related embodiments, the invention provides such methods that further comprise isolating and cloning the cell before introducing it into the eukaryote. As used herein, the phrases "conditions favoring the production" of an expression product, "conditions favoring overexpression" of a gene, and "conditions favoring the activation" of a gene, in a cell or by an in vitro cell refers to any and all environmental, physical, nutritional or biochemical parameters that allow, facilitate, or promote the production of an expression product, or overexpression or activation of a gene, through an in vitro cell. Of course, said conditions may include the use of culture media, incubation, delivery, moisture, etc., which are optimal or which allow, facilitate, or promote the production of an expression product, or overexpression or activation of a gene, by an in vitro cell. Analogously, as used herein, the phrases "conditions favoring the production" of an expression product, "conditions favoring overexpression" of a gene, in a cell or by an in vivo cell refer to any and all the environmental, physical, nutritional, biochemical, behavioral, genetic and emotional parameters under which an animal that contains a cell is maintained, which allows, facilitates or promotes the production of an expression product, or overexpression or activation of a gene , by a cell in a eukaryotic in vivo. A person skilled in the art can determine whether a given set of conditions is favorable for the expression, activation, or overexpression of the gene in vitro or in vivo, using the selection methods described and exemplified below, or using other methods for measure the expression, activation or overexpression of the gene, which are already routine in the art. The invention also encompasses cells obtained by any of the above methods. The invention encompasses cells containing the vector constructs, cells in which the vector constructs have been integrated, and cells overexpressing the desired gene products from an endogenous gene, said overexpression has been driven by the introduced transcriptional regulatory sequence. . The cells used in this invention can be derived from any eukaryotic species and can be primary, secondary or immortalized. Moreover, cells can be derived from any tissue in the body. Examples of useful tissues from which the cells can be isolated and activated include, but are not limited to, liver, kidney, bladder, bone marrow, thymus, heart, muscle, lung, brain, testis, ovary, islet, intestinal cells , skin, bone, gallbladder, prostate, vesicle, embryos, and hematopoietic immune systems. Cell types include fibroblastic, epithelial, neuronal, stem, and follicular cells. However, any cell or cell type can be used to activate the expression of the gene that is used in this invention. The methods that can be carried out in any of the cells of eukaryotic origin, such as fungal, vegetable or animal. Preferred embodiments include vertebrates and particularly mammals, most particularly humans.

The construction can be integrated into primary, secondary or immortalized cells. The primary cells are cells that have been isolated from a vertebrate and that have not been transferred. Secondary cells are cells that have been crossed, but not immortalized, immortalized cells are cell lines that can apparently be traded indefinitely. In preferred embodiments, the cells are immortalized cell lines. Examples of immortalized cell lines include, but are not limited to, HT1080, HeLa, Jurkat, 293 cells, KB carcinoma, T84 colonic epithelial cell line, Raji, Hep G2 or Hep 3B hepatic cell lines, A2058 melanoma, lymphoma. U937, and WI38 fibroblast cell line, somatic cell hybrids, and hybridomas. The cells used in this invention can be derived from any eukaryotic species, including but not limited to mammary cells (such as cells from rats, mice, cattle, swine, sheep, goats and humans) bird cells, fish cells, amphibian cells, reptile cells, plant cells, and yeast cells. Preferably, overexpression of an endogenous gene or gene product of particular species is achieved by activating gene expression in a cell of those species. For example, human cells are used to overexpress endogenous human proteins. Similarly, bovine cells are used to overexpress endogenous bovine proteins, for example bovine growth hormone.

The cells can be derived from any tissue in the eukaryotic organism. Examples of useful vertebrate tissues from which the cells can be isolated and activated include, but are not limited to, liver, kidney, bladder, bone marrow, thymus, heart, muscle, lung, brain, immune system (including lymphatic) cells. , testicles, ovaries, islets, intestines, stomachs, bone marrow, skin, bone, bladder, gallbladder, prostate, gallbladder, zygote, embryos and hematopoietic tissue. Useful vertebrate cell types include, but are not limited to, fibroblasts, epithelial cells, neuronal cells, germ cells (ie, spermatocytes / sperm and voids), stem cells, and follicular cells. Examples of plant tissues from which the cells can be isolated and activated include, but are not limited to, leaf tissues, ovarian tissues, stamen fabrics, pistil tissues, root tissues, tuber tissues, gametes, seeds, embryos and the like. Those skilled in the art will appreciate, however, that any eukaryotic cell or cell type can be used to activate the gene expression utilizing the present invention. Any of the cells produced by any of the described methods are useful for selecting the expression of a desired gene product and for providing desired amounts of a gene product that is overexpressed in the cell. The cells can be isolated and cloned. The cells produced by this method can be used to produce the protein in vitro (for example, for use as a protein therapy) or in vivo (for example, for use in cell therapy). Commercial growth and production conditions frequently vary from the conditions used to grow and prepare cells for analytical use (eg, cloning, protein or nucleic acid sequencing, elevation antibodies, X-ray crystallography analysis, Enzymatic analysis, and the like The method for enlarging the cells in proportion to obtain their production in roller bottles involves the increase in the surface area in which the cells can bind, therefore microcarrier spheres are frequently added to increase the area of surface for commercial production The method for enlarging cells in proportion in rotating crops may involve increases in volume.Five liters or more may be required for the microcarrier for production by rotary methods.Depending on the inherent potency (specific activity) of the protein of interest, the volume can be low as 1-10 liters. 10-15 liters is more common. However, up to 50-100 liters may be necessary and the volume may be as high as 10,000 to 15,000 liters. In some cases higher volumes may be required. The cells can also be produced in large quantities of T flasks, for example from 50 to 100. Despite the production conditions, the purification of protein on a commercial scale can also vary considerably from the purification for analytical purposes. The purification of proteins in a commercial practical context can initially be the mass equivalent of 10 liters of cells approximately 104 cells / ml. The cell mass equivalent to initiate protein purification can be as high as 10 liters of cells up to 10 6 or 10 7 cells / ml. However, as one skilled in the art will appreciate, a lower or higher initial cellular mass equivalent may be of great use and of great advantage so that it can be conveniently used in these methods. Another condition of commercial production, especially when the final product is used clinically, is the cellular production in a serum-free medium, by means of which the medium is intended to contain no serum or that does not contain it in quantities required for cell production. This obviously avoids unwanted co-purification of toxic contaminants (e.g., viruses) or other types of contaminants, e.g., proteins that would complicate purification. The serum-free media for cell production, commercial sources for such media, and methods for cell cultures in serum-free media, are known to those skilled in the art. A simple cell obtained by these methods described above can overexpress a single gene or more than one gene. More than one gene can be activated by integrating a simple construction or by integrating multiple constructions into the same cell (ie, more than one type of construction). Therefore, a cell can contain only one type of vector construct or different types of constructions, and each can activate an endogenous gene. The invention is also directed to methods for obtaining the cells described above by one or more of the following steps: introducing one or more of the vector constructs; allowing the introduced constructs to be integrated into the cell genome by non-homologous recombination, allowing the overexpression of one or more endogenous genes in the cell; as well as isolate and clone the cell. The term "transfection" has been used herein for convenience, when describing the introduction of a polynucleotide into a cell. However, it should be understood that the specific use of this term has been applied to generally refer to the introduction of the polynucleotide into a cell and is also intended to refer to the introduction by other methods described herein such as electroporation, mediated introduction by liposome, retrovirus-mediated introduction, and the like (as well as according to this specific meaning). The vector can be introduced into the cell by a number of methods known in the art. These include, but are not limited to, electroporation, calcium phosphate precipitation, DEAE dextran, lipofection, and receptor-mediated endocytosis, polybrene, particle bombardment, and microinjection. Alternatively, the vector can be delivered to the cell as a viral particle (either competent or deficient replication). Examples of viruses useful for the delivery of nucleic acid include, but are not limited to, adenoviruses, adeno-associated viruses, retroviruses, herpes viruses, and vaccine viruses. Other viruses suitable for the delivery of nucleic acid molecules in cells are known to those skilled in the art and can be used equivalently in current methods. After transfection, the cells are cultured under conditions, as are known in the art, suitable for non-homologous integration between the vector and the host cell genome. Cells that contain the integrated vector in a non-homologous manner can be further cultured under conditions, which are already known in the art, that allow the expression of activated endogenous genes. The vector construct can be introduced into cells in a simple DNA construct or in separate constructions and allowed to concatemerize. Considering the preferred embodiments, the vector construct is a double-stranded DNA vector construct, the vector constructs also include single-stranded DNA, single-stranded and double-stranded DNA combinations, single-stranded RNA, double-stranded RNA chain, and combinations of single chain and double chain RNA. Therefore, as an example, the vector construct could be single-stranded RNA that is converted to cDNA by means of reverse transcriptase, the cDNA converted to double-stranded DNA, and the double-stranded DNA finally recombine with the genome of host cell.

In preferred embodiments, the constructs are linearized prior to introduction into the cell. Linearization of the activation construct generates free DNA ends capable of reacting with chromosomal ends during the integration procedure. In general, the construction is linearized downstream of the regulatory element (and the exonic and splice donor sequences, if present). Linearization can be facilitated by, for example, the placement of a single restriction site downstream of the regulatory sequences and the treatment of the construction with the corresponding restriction enzyme prior to transfection. As long as it is not required, it is convenient to place a "separator" sequence between the linearization site and the more functional nearby element (for example, the unpaired splice donor site) in the construction. When the spacer sequence is present, it protects the important functional elements in the vector of exonucleic degradation during the transfection process. The spacer sequence can be composed of any nucleotide sequence that does not change the essential functions of the vector as described herein. The circular constructions can also be used to activate the expression of the endogenous gene. Circular plasmids, as is known in the art, at the time of transfection in cells, can be integrated into the genome of the host cell. Probably, DNA breaks occur in the circular plasmid during the transfection procedure, generating there free DNA ends capable of binding to the chromosomal ends. Some of these construction breaks will occur in a position that does not destroy the essential functions of the vector (for example, the breakdown will occur downstream of the regulatory sequence), and therefore, allow the construction to be integrated into a chromosome in a configuration that can activate an endogenous gene. As described above, the separator sequences can be placed in the construct (eg, downstream of the regulatory sequences). During transfection, cleavages occurring in the separating region will generate free ends at the site in the proper construction for the activation of an endogenous gene after integration into the host cell genome. The invention also encompasses libraries of cells formed by the methods described above. A library can encompass all clones of a simple transfection experiment or encompass a subset of clones from a simple transfection experiment. The subgroup may overexpress the same gene or more than one gene, for example, a class of genes. The transfection could have been carried out with a simple type of construction or with more than one type of construction. A library can also be formed by combining all recombinant cells from two or more transfection experiments, by combining one or more subgroups of cells from a single transfection experiment or by combining cell subgroups from transfection experiments carried out separately. The resulting library can express the same gene, or more than one gene, for example a class of genes. Again, in each of these individual transfections, a single construction or more than one construction can be used. The libraries can be formed from the same cell type or from different cell types. The library can be composed of a single cell type that contains a unique type of activation construct that has been integrated into the chromosomes in spontaneous DNA breaks or in breaks generated by radiation, restriction enzymes, and / or agents of DNA cleavage, applied either bound (to the same cells) or separately (applied to individual groups of cells and then combining the cells together to produce the library). The library can be composed of multiple cell types that contain single or multiple constructs that were integrated into the genome of a cell treated with radiation, restriction enzymes, and / or DNA breaking agents, applied either attached (to the same cells) or separately (applied to individual groups of cells and then combining the cells together to produce the library). The invention also relates to methods for forming libraries by selecting several subgroups of cells from the same or different transfection experiments. For example, all cells expressing nuclear factors (determined by the presence of green fluorescent nuclear protein in cells transfected with construct 20) can be pooled to generate a cell library with activated nuclear factors. Similarly, cells expressing membrane or secreted proteins can be pooled. The cells can also be grouped by phenotype, eg growth factor independent growth, growth factor-independent proliferation, colony formation, cell differentiation (eg, differentiation within a neuronal cell, muscle cell, epithelial cell, etc.). ), anchoring independent growth, activation of cellular factors (eg, kinases, transcription factors, nucleases, etc.), gain or loss of cell-to-cell adhesion, migration, or cellular activation (eg, resting T cells against activated T cells). The invention further relates to methods for using libraries of cells that overexpress an endogenous gene. The library is selected for the expression of the gene and the cells are selected to express the desired gene product. The cell can then be used to purify the gene product for subsequent use. Cell expression can occur when the cell is cultured in vitro or by allowing the cell to express the gene in vivo. The invention also relates to methods for using libraries and thus identifying the novel gene and the gene products. The invention also relates to methods for increasing the efficiency of gene activation by treating the cells with agents that stimulate or cause an effect on non-homologous integration patterns. It has been shown that gene expression patterns, chromatin structure and methylation patterns can differ dramatically from one type of cell to another type of cell. Even different cell lines of the same cell type can have significant differences. These differences can cause an impact on non-homologous integration patterns by affecting both the DNA breakdown pattern and the repair procedure. For example, chromatized DNA spaces (characteristics likely associated with inactive genes) may be more resistant to breakage by restriction enzymes and chemical agents, considering that they may be susceptible to radiation breakage. In addition, inactive genes can be methylated. In this case, restriction enzymes that are blocked by CpG methylation will be unable to cut methylated sites near the inactive gene making it more difficult to activate that gene using enzymes sensitive to methylation. These problems can be counteracted by generating activation libraries in different cell lines using a variety of DNA breaking agents. By carrying out the above, a more complete integration pattern can be generated and the probability of activating a given gene can be maximized. The methods of the invention may include the introduction of double-strand breaks in the DNA of the cell containing the endogenous gene to be overexpressed. These methods exhibit double-strand breaks in the genomic DNA in the cell prior to or simultaneously with the integration of the vector. The mechanism of DNA breakdown can have a significant effect on the pattern of DNA breaks in the genome. As a result, DNA breaks produced spontaneously or artificially with radiation, restriction enzymes, bleomycin, or other breaking agents, can occur in different positions. To increase the integration efficiency and thus improve the random distribution of integration sites, cells can be treated with low, intermediate, or high doses of radiation prior to or after transfection. By the artificial induction of double-strand breaks, the transfected DNA can now be integrated into the chromosome of the host cell as part of the DNA repair procedure. Normally, the generation of double-stranded breaks that function as the integration site is the limiting frequency stage. Therefore, by increasing chromosomal breaks using radiation (or other agents that damage DNA), a large number of members can be obtained in a given transfection. Moreover, the mechanism of breaking DNA by radiation is different from the mechanism by spontaneous breaking. Radiation can include DNA breaks directly when a high-energy photon collides with the DNA molecule. Alternatively, radiation can activate compounds in the cell that in turn react with and break the DNA strand. On the other hand, it is considered that spontaneous breaks occur due to the interaction between the reactive compounds produced in the cell (such as superoxides and peroxides) and the DNA molecule. However, the DNA in the cell is not present as a naked polymer, deproteneized, but instead, it is bound to the chromatin and present in a condensed state. As a result, some regions are not accessible to agents in the cell that cause double-stranded breaks. Photons produced by radiation have wavelengths short enough to collide with highly condensed regions of DNA, thus inducing breaks in the regions of DNA that are represented below in spontaneous breaks. Therefore, radiation can generate different DNA breaking patterns, which in turn, should lead to different patterns of integration. As a result, libraries produced using the same activation construct in cells with or without radiation treatment will potentially contain different groups of activated genes. Finally, radiation treatment increases the non-homologous integration efficiency up to 5 to 10 times, allowing complete libraries to be generated using a smaller number of cells. Therefore, radiation treatment increases the efficiency of gene activation and generates new patterns of integration and activation in the transfected cells. Useful types of radiation include radiation with rays, ß,?, X and ultraviolet radiation. Useful doses of radiation vary for different cell types, but in general, dose scales that result in cell viabilities of 0.1% a >are useful.99% For HT1080 cells, this corresponds to radiation doses from a 137Cs source from about 0.1 rads to 100 rads. Other doses may also be useful, as long as the dose increases the frequency of integration or changes the pattern of integration sites. In addition to radiation, restriction enzymes can be used to artificially induce chromosomal breaks in transfected cells. As with radiation, DNA restriction enzymes can generate chromosomal breaks which, in turn, function as the integration sites for the transfected DNA. This large number of DNA breaks increases the overall integration efficiency of the activation construct. Additionally, the mechanism of cleavage by restriction enzymes differs from the mechanism by radiation, and the pattern of chromosomal breaks is likely to be different. Restriction enzymes are relatively large molecules compared to photons and smaller metabolites capable of damaging DNA. As a result, restriction enzymes will tend to break regions that are less condensed than the genome considered as a whole. If the gene of interest is within an accessible region of the genome, then treatment of the cells with a restriction enzyme may increase the likelihood of integrating the activation construct upstream of the gene of interest. Since restriction enzymes recognize specific sequences, and as a given restriction site may not be upstream of the gene of interest, a variety of restriction enzymes may be used. It may also be important to use a variety of restriction enzymes since each enzyme has different properties (for example: size, stability, ability to cut methylated sites, and optimal reaction conditions) that affect the determination of which sites on the chromosome host that will be cut. Each enzyme, due to the different distribution of restriction sites susceptible to cutting, will generate a different integration pattern. Therefore, the introduction of restriction enzymes (or plasmids capable of expressing for restriction enzymes), before, during, or after the introduction of the activation construct will result in the activation of different groups of genes. Finally, cleavage induced by restriction enzymes increases the integration efficiency up to 5 to 10 times (Yorifuji er al., Mut. Res 243: 121 (1990)), allowing a smaller number of cells to be transfected to produce a complete library . Therefore, restriction enzymes can be used to generate new integration patterns, allowing the activation of genes that were not activated in libraries produced by non-homologous recombination in spontaneous breaks or in other artificially induced breaks. Restriction enzymes can also be used to shift the integration of the activation construct to a desired site in the genome. For example, various rare restriction enzymes have been described that cut eukaryotic DNA every 50 to 1000 kilobases, on average. If it happens that a rare restriction recognition sequence is located upstream of a gene of interest, by introducing the restriction enzyme at the time of transfection together with the activation construct, the DNA breaks may preferably be upstream of the interest. These breaks can then function as sites for the integration of the activation construction. It may be that any enzyme cuts into an appropriate position in or near the gene of interest and its site is down represented in the rest of the genome or its site is overrepresented near the genes (eg, restriction sites containing CpG). For genes that have not been previously identified, restriction enzymes can be used with 8bp recognition sites (eg, Not \, S / 7I, Pmel, Swa \, Sse \, SrfL, SgrA \, Pac, Asc \, Sg / \, and Sse83871), enzymes that recognize sites that contain CpG (eg, Eagl, Bsi-WI, Mlu \, and SssHIl) and other rare-cut enzymes. In this way, "deviated" libraries can be generated, which are enriched for certain types of activated genes. In this regard, restriction enzyme sites containing CpG dinucleotides are particularly useful, since these sites are underrepresented in the genome over their length, but overrepresented in the form of CpG islands at the 5 'end of many genes, the position which is very useful for the activation of the gene. Accordingly, enzymes that recognize these sites will preferably cut at the 5 'end of the gene sequences. Restriction enzymes can be introduced into the host cell by various methods. First, restriction enzymes can be introduced into the cell by electroporation (Yorifuji et al., Mut Res 243: 121 (1990); Winegar et al., Mut. Res 225: 49 (1989)). In general, the amount of restriction enzyme introduced into the cell is proportional to its concentration in the electroporation medium. The pulse conditions must be optimized for each cell line by adjusting the voltage, capacitance and resistance. Second, the restriction enzyme can be transiently expressed from a plasmid encoding the enzyme under the control of eukaryotic regulatory elements. The level of enzyme produced can be controlled using inducible promoters, and varying the strength of induction. In some cases, it would be desirable to limit the amount of restriction enzyme produced (due to its toxicity). In these cases, weak or mutant promoters, splice sites, translation start codons, and poly A ends can be used to decrease the amount of restriction enzyme produced. Third, restriction enzymes can be introduced by agents that fuse with or permeabilize the cell membrane. Liposomes and streptolysin O (Pimplikar et al., J. Cell Biol. 725: 1025 (1994)) are examples of this type of agent. Finally, mechanical drilling can also be used (Beckers et al., Cell 50: 523-534 (1987)) and microinjection to introduce nucleases and other proteins into the cells. However, any method that can supply active enzymes to a living cell is suitable. DNA breaks induced by bleomycin and other DNA-damaging agents can also produce DNA breaking patterns that are different. Therefore, any agent or incubation condition that has the ability to generate double-stranded breaks in the cells, is useful for increasing the efficiency and / or altering non-homologous recombination sites. Examples of classes of chemical DNA breaking agents include, but are not limited to, peroxides and other free radical generating compounds, alkylating agents, topoisomerase inhibitors, antineoplastic drugs, acids, substituted nucleotides and enediin antibiotics. Specific chemical DNA breaking agents include, but are not limited to, bleomycin, hydrogen peroxide, cumene hydroperoxide, tert-butyl hydroperoxide, hypochlorous acid (reacted with aniline, -1-naphthylamine or 1-naphthol), nitric acid, phosphoric acid, doxorubicin, 9-deoxidoxorubicin, demethyl-6-deoxirubicin, 5-iminodaunorubicin, adriamycin, 4 '- (- 9-acridinylamino) methanesulfon-m-anisidide, neocarzinostatin, 8-methoxycaffeine, etoposide, ellipticine, iododeoxyuridine and bromodeoxidiuridine. It has been shown that the DNA repair machinery in the cell can be induced by prior exposure of the cell at low doses of a DNA breaking agent such as radiation or bleomycin. By pre-treating cells with these agents for approximately 24 hours prior to transfection, the cell will be more efficient in repairing DNA breaks and integrating the DNA after transfection. In addition, higher doses of radiation or other DNA breaking agents can be used since the LD50 (the dose causes mortality in 50% of the exposed cells) is higher after the previous treatment. This allows randomized activation of libraries at multiple doses and results in a different distribution of integration sites within the chromosomes of the host cell.

Selection Once an activation library (or libraries) has been generated, you can select it (s) by performing a number of tests. Depending on the characteristics of the protein (s) of interest (e.g., proteins secreted against intracellular proteins) and the nature of the activation construct used to generate the library, any or all of the tests described below may be used. . Other test formats can also be used.

ELISA- Activated proteins can be detected using the enzyme-linked immunosorbent assay (ELISA). If the activated gene product is secreted, the culture supernatants of the activation gene cell groups are incubated in wells containing a bound antibody specific for the protein of interest. If a cell or group of cells has activated the gene of interest, then the protein will be secreted into the culture medium. By selecting groups of clones from a library (groups can be from 1 to greater than 100,000 elements of the library), one can identify the groups that contain a cell (s) that have (n) activated the gene of interest. The cell of interest can then be purified from other library members by "sib" inbreeding selection, limiting dilution, or other methods known in the art. In addition to the secreted proteins, the ELISA test can be used to select cells that express intracellular proteins and membrane-bound proteins. In these cases, instead of selecting culture supernatants, a small number of cells are removed from the library group (each cell is represented at least 100 to 1000 times in each group), used, clarified, and added to the coated wells with antibodies.

ELISA test by drip. The ELISA test drops are coated with antibodies specific for the protein of interest. After coating, the wells are blocked with 1% BSA / PBS for one hour at 37 ° C. After blockade, 100,000 to 500,000 cells of the randomly activated library are applied to each well (this represents ~ 10% of the total group.

Generally, a group is applied to each well. If the frequency of a cell expressing the protein of interest is 1 in 10,000 (ie, the group consists of 10,000 individual clones, each of which expresses the protein of interest), then plating 500,000 cells per well , will yield 50 specific cells. The cells are incubated in the wells at 37 ° C for 24 to 48 hours without being moved or disturbed. At the end of the incubation, the cells are removed and the plate is washed 3 times with PBS / 0.05% Tween 20 and 3 times with PBS / 1% / BSA. Secondary antibodies are applied to the wells in the proper concentration and are incubated for two hours at room temperature or 16 hours at 4 ° C. These antibodies can be biotinylated or directly labeled with horseradish peroxidase (HRP). The secondary antibodies are removed and the plate is washed with PBS / 1% / BSA. The tertiary antibody or streptavidin labeled with HRP is added and incubated for one hour at room temperature.

Test FACS. The fluorescence activated cell sorter (FACS) can be used to select the random activation library in various forms. If the gene of interest codes for a cell surface protein, then the fluorescently labeled antibodies are incubated with cells from the activation library. If the gene of interest codes for a secreted protein, then the cells can be biotinylated and incubated with streptavidin conjugated to an antibody specific for the protein of interest (Manz et al., Proc. Nati. Acad Sci. (USA) 92: 1921 (nineteen ninety five)). After incubation, the cells are placed in a high concentration of gelatin (or another polymer, such as agarose or methylcellulose) to limit the diffusion of the secreted protein. As the protein is secreted by the cell, this is captured by the antibody bound to the cell surface. The presence of the protein of interest is then detected by a second antibody that is fluorescently labeled. The cells can be classified according to their fluorescence signal, both for the secreted proteins and for the membrane-bound proteins. The fluorescent cells can then be isolated, expanded, and then enriched by FACS, limiting dilution, or other cellular purification teches known in the art.

Separation of magnetic spheres. The principle of this technique is similar to that of FACS. The membrane bound proteins and the captured secreted proteins (as described above) are detected by incubating the activation library with magnetic beads conjugated with antibody, which are specific for the protein of interest. If the protein is present on the surface of a cell, the magnetic spheres will bind to that cell. Using a magnet, cells expressing the protein of interest can be purified from other cells in the library. The cells are then released from the spheres, expanded, analyzed and subsequently purified if necessary.

RT-PCR. A small number of cells (equivalent to at least the number of individual clones in the group) is cultured and lysed to allow RNA purification. After isolation, the RNA is reverse transcribed using reverse transcriptase. PCR is then carried out using specific primers for cDNA of the gene of interest. Alternatively, primers encompassing the synthetic exon in the activation construct and the exon of the endogenous gene can be used. This primer will not hybridize or amplify the gene of interest expressed endogenously. On the contrary, if the activation construct has been integrated upstream to the gene of interest and has activated the expression of the gene, then this primer, together with a second specific primer for the gene will amplify the activated gene due to the presence of the synthetic exon spliced on the exon of the endogenous gene. Therefore, this method can be used to detect activated genes in cells that normally express the gene of interest at lower levels than desired.

Phenotypic Section In this embodiment, the cells can be selected based on a phenotype conferred by the activated gene. Examples of phenotypes that can be selected are: proliferation, growth factor-independent growth, colony formation, cell differentiation (for example, differentiation in a neuronal cell, muscle cell, epithelial cell, etc.), independent anchoring growth, activation of cellular factors (e.g., kinases, transcription factors, nucleases, etc.), gain or loss of cell-to-cell adhesion, cell migration and activation (e.g., resting T cells against activated T cells). It is important to isolate activated cells that demonstrate a phenotype, such as that described above, due to the activation of an endogenous gene by integrated construction and is probably responsible for the observed cell phenotype. Therefore, the activated gene can be a therapeutic drug or important drug target to treat or induce the observed phenotype. The sensitivity of each of the above tests can be increased effectively by transiently overregulating the expression of the gene in the cells of the library. This can be achieved for promoters containing NF-βB on site (in the activation construct) by adding PMA and tumor necrosis factor-a, eg, to the library. Separately, or in conjunction with PMA and TNF-a, sodium butyrate can be added to further increase gene expression. The addition of these reagents can increase the expression of the protein of interest, thus giving rise to a lower sensitivity test which is used to identify the activated gene of the cell of interest. As the major activation libraries are generated to maximize the activation of a large number of genes, it is a great advantage to organize the clones of the library into groups. Each group can consist of 1 to more than 100,000 individual clones. Therefore, in a given group, many activated proteins are produced, often in diluted concentrations (due to the overall size of the group and the limited number of cells within the group)., which produce a certain activated protein). Therefore, the concentration of proteins before selection effectively increases the ability to detect activated proteins in the selection test. A particularly useful method of concentration is ultrafiltration. However, other methods can be used. For example, proteins can be concentrated non-specifically, or semi-specifically by adsorption on the exchange of ions, hydrophobic resins, dyes, hydroxyapatite, lectin other suitable resins under conditions that bind most of all proteins present The bound proteins can then be removed in a small volume before selection. It is a great advantage to grow the cells in a serum-free medium to facilitate the concentration of proteins. In another embodiment, a sequence useful in the activation construct, which is an epitope tag, may be included. The epitope tag consists of an amino acid sequence that allows for affinity purification of the activated protein (e.g., in immunoaffinity or chelating matrices). Therefore, by including an epitope tag in the activation construct, all activated proteins from an activation library can be purified. By purifying activated proteins from other proteins cellular media, selection for novel proteins enzymatic activities can be facilitated. In some cases, it would be desirable to remove the epitope tag after purification of the activated protein. This can be achieved by including a protease recognition sequence (e.g., Factor Ha or enterokinase cleavage site) downstream of the epitope tag in the activation construct. Incubation of the purified protein (s) purified with the appropriate protease will release the epitope tag of the protein (s). In those libraries in which a sequence of epitope tags is located in the activation construct, all activated proteins can be purified from all other proteins cell media using affinity purification. This not only concentrates the activated proteins, but also purifies them from other activities that may interfere with the test used to select the library. Once a clone group containing cells that overexpress the gene of interest is identified, some steps can be taken to isolate the activated cell. The isolation of the activated cell can be achieved by a variety of methods known in the art. Examples of cell purification methods include limiting dilution, selection of fluorescence activated cells, separation of magnetic spheres, "sib" inbreeding selection purification of single colonies using cloning rings.

In preferred embodiments of the invention, the methods include a method wherein the expression product is purified. In highly preferred embodiments, the cells expressing the endogenous gene product are cultured in such a way as to produce quantities of the gene product that is feasible for commercial application, and especially for various uses such as diagnosis and therapeutic and drug discovery. Any vector used in the methods described herein may include an amplifiable marker. Therefore, the amplification of both the vector and the DNA of interest (ie, which contains the overexpressed gene) occurs in the cell, and the additional increased expression of the endogenous gene is obtained. In accordance with the foregoing, the methods may include a step in which the endogenous gene is amplified. Once the activated cell has been isolated, the expression can be further increased by expanding the locus that contains both the gene of interest and the activation construct. This can be achieved by each of the methods described below, either separately or in combination. Amplifiable markers are genes that can be selected for a greater number of copies. Examples of amplifiable labels include dihydrofolate reductase, adenosine deaminase, aspartate transcarbamylase, dihydro-orotase and carbamyl phosphate synthase. For these examples, the high number of copies of the amplifiable marker and of the flanking sequences (including the gene of interest) can be selected so that a toxic drug or metabolite acting on the amplifiable marker is used. In general, as the concentration of the drug or toxic metabolite increases, cells containing a lower number copies of the amplifiable marker die, while cells containing increased copies of the marker survive and form colonies. These colonies can be isolated, expanded and analyzed for increased levels of production of the gene of interest. The placement of an amplifiable marker in the activation construct results in the juxtaposition of the gene of interest and the amplifiable marker in the activated cell. The selection for activated cells containing an increased number of copies of the amplifiable marker and the gene of interest can be achieved by cell growth or cell culture in the presence of increased amounts of a selective agent (generally a drug or metabolite). For example, the amplification of dihydrofolate reductase (DHFR) can be selected using metrotrexate. As the drug-resistant colonies arise at each increased concentration of the drug, the individual colonies can be selected and characterized for the number of copies of the amplifiable marker and the gene of interest, and analyzed for the expression of the gene of interest. Individual colonies with the highest levels of expression of the activated gene can be selected to achieve additional amplification at higher drug concentrations. At the highest drug concentrations, the clones will express considey increased amounts of the protein of interest. When DHFR is amplified, it is convenient to plague approximately 1 x 107 cells at different concentrations of methotrexate. The useful initial methotrexate concentrations range from about 5 nM to 100 nM. However, the optimal concentration of metrotrexate must be determined empirically for each cell line and for each site of integration. After growth in the medium containing methotrexate, the colonies of the highest concentration of methotrexate are chosen and analyzed for the increased expression of the gene of interest. The clone (s) with the highest concentration of methotrexate are then grown in higher concentrations of methotrexate to select the additional amplification of DHFR and the gene of interest. Methotrexate concentrations on the micromolar and millimolar scale can be used for clones that contain the highest degree of gene amplification. The placement of a viral origin of replication (s) (for example, ori P or SV40 in human cells, and polyoma ori in mouse cells) in the activation construct will cause the juxtaposition of the gene of interest and the viral origin of replication in the activated cell. The sequences of origin and flanking can then be amplified by introducing the viral replication proteins in trans. For example, when ori P (the origin of replication in the Epstein-Barr virus) is used, EBNA-I can be expressed transiently or stably. EBNA-I will initiate the replication of the integrated ori P locus. The replication will be bi-directionally extended from the origin. As each replication product is generated, replication can also begin. As a result, many copies of the sequences of viral origin and genomic flanking sequences including the gene of interest are generated. This higher number of copies allows the cells to produce larger quantities of the gene of interest. At some frequency, the replication product will recombine to form a circular molecule containing genomic flanking sequences, including the gene of interest. Cells containing circular molecules with the gene of interest can be isolated by simple cell cloning and by analysis by Hirt extraction and Southern blotting. Once purified, the cell containing the episomal genomic locus in the high number of copies (typically 10-50 copies) can be propagated in the culture. In order to achieve greater amplification, the episome can also be elevated by including a second origin adjacent to the first in the original construction. For example, T antigen can be used to raise the number of copies of episomes from ori P / SV40 to a copy number of -1000 (Heinzel et al., J. Virol. 62: 3738 (1988)). substantial in the number of copies can dramatically increase the expression of the protein. The invention contemplates the overexpression of endogenous genes both in vivo and in vitro. Accordingly, the in? Ior cells could be used to produce desired amounts of a gene product or they could be used in vivo to provide the gene product in the intact animal. The invention also contemplates the proteins produced by the methods described herein. The proteins can be produced either from known genes, or from previously unknown genes. Examples of known proteins that can be produced by this method include, but are not limited to, erythropoietin, insulin, growth hormone, glycocerebrosidase, tissue plasminogen activator, granulocyte colony stimulating factor, granulocyte / macrophage colony stimulating factor, interferon a, interferon ß, interferon,, interleukin-2, interleukin-6, interleukin-11, interleukin-12, TGF ß, blood coagulation factor V, blood coagulation factor VII, blood coagulation factor VIII, blood coagulation factor IX, blood coagulation factor X, TSH-β, bone growth factor 2, bone growth factor-7, tumor necrosis factor, alpha-1-antitrypsin, anti-thrombin III, inhibitory factor of leukemia, glucagon, protein C, protein kinase C, macrophage colony stimulating factor, stem cell factor, follicle stimulating hormone urokinase, growth factors d e nerves, growth factors in the form of insulin, insulinotropin, parathyroid hormone, lactoferrin, complement inhibitors, growth factor derived from platelets, keratinocyte growth factor, neurotropin-3, thrombopoietin, chorionic gonadotropin, thrombomodulin, alpha glucosidase, factor of epidermal growth, FGF, macrophage colony stimulating factor and cell surface receptors for each of the proteins described above. When the protein product of the activated cell is purified, any method of protein purification known in the art can be used.

Isolation of cells containing activated genes that code for membrane protein. The genes that encode membrane-associated proteins are particularly interesting from the point of view of drug development. These genes, and the proteins they encode, can be used, for example, to develop small molecule drugs using combinatorial chemistry libraries and high throughput screening tests. Alternatively, proteins or soluble forms of the proteins (eg, truncated proteins lacking the transmembrane region) can be used as the therapeutically active agents in humans or animals. Identification of membrane proteins can also be used to identify new ligands (eg, cytokines, growth factors and other effector molecules) using two hybrid approaches or affinity capture techniques. Other diverse uses of membrane proteins are also possible. Current approaches for identifying genes encoding integral membrane proteins encompass the isolation and sequencing of genes from cDNA libraries. The integral membrane proteins are identified by ORF analysis (open reading frame) using hydrophobic capacity plots that can identify the transmembrane region of the protein. Unfortunately, by using this approach, a gene encoding an integral membrane protein can not be identified, unless the gene is expressed in the cells used to produce the cDNA library. Moreover, many genes are only expressed in rare cells, during reduced experimental windows, and / or at other very low levels. As a result, these genes can not be identified efficiently using the currently available approaches. The present invention allows endogenous genes to be activated without any knowledge of the sequence, structure, function, or expression profile of the genes. Using the described methods, the genes can be activated at the transcription level only, or at both levels, both transcription and translation. As a result, the proteins encoded by the activated endogenous gene can be produced in cells containing the integrated vector. Moreover, by using the specific vectors described herein, the protein produced from the activated endogenous gene can be modified, for example, to include an epitope tag. Other vectors, (e.g., vectors 12-17 described above) can encode a signal peptide after an epitope tag. This vector can be used to isolate cells that have activated the expression of an integral membrane protein (see, Example 5 below). This vector can also be used to select the secretion of proteins that are not normally secreted. Therefore, the invention also relates to methods for identifying an endogenous gene that encodes an integral cell membrane protein or a transmembrane protein. Said methods of the invention may comprise one or more steps. For example, such a method of the invention may comprise: a) introducing one or more vectors of the invention into a cell; b) allow the vector to integrate into the cell genome by non-homologous recombination; c) allowing overexpression of an endogenous gene in the cell by overregulating the gene by the transcription regulatory sequence that is contained in the integrated vector construct; c) selecting the cell for overexpression of the endogenous gene; and e) characterizing the activated gene to determine its identity as a gene encoding an integral cellular membrane protein. In related embodiments, the invention provides such methods that further comprise isolating the activated gene from the cell prior to characterization of the activated gene. To identify genes that encode integral membrane proteins, the vectors integrated into the cell genome will comprise a regulatory sequence linked to an exonic sequence containing a start codon, a signal sequence and an epitope tag, followed by an unpaired splice donor site. When the integration and activation of an endogenous gene is carried out, a chimeric protein is produced which contains the signal peptide and the epitope tag of the vector fused to the protein, encoded by the exons downstream of the endogenous gene. This chimeric protein, by virtue of the presence of the vector encoded signal peptide, is directed to the secretory pathway where the translation of the protein is terminated and the protein is secreted. However, if the activated endogenous gene encodes an integral membrane protein, and the transmembrane region of that gene is encoded by exons located 3 'from the vector's integration site, then the chimeric protein will be driven to the cell surface, and the epitope tag will be displayed on the cell surface. Using known methods of cell isolation (eg, flow cytometric classification, cell selection by magnetic spheres, immunoadsorption, or other methods that will be familiar to those skilled in the art), antibodies to the epitope tag can then be used. to isolate cells from the population that shows the epitope tag and that have activated a gene that encodes the integral membrane. These cells can then be used to study the function of the membrane protein. Alternatively, the activated gene can then be isolated from these cells using any method known in the art, for example by hybridization with a DNA probe specific for the vector-encoded exon to select a cDNA library produced from these cells , or using the genetic constructions described herein. The epitope tag encoded by the vector exon can be a minor peptide that can bind to an antibody, a minor peptide that can bind to a substance (eg, polyhistidine / divalent metal ion supports, protein supports linked to maltose / maltose supports, glutathione-S-transferase / glutathione support), or an extracellular domain (lacking a transmembrane domain) of an integral membrane protein for which an antibody or ligand exists. However, it will be understood that other types of epitope marks that are familiar to those skilled in the art may be used in an equivalent manner according to the invention. Other modifications and adaptations suitable to the methods and applications described herein, will be readily apparent to those skilled in the relevant arts and may be carried out without departing from the spirit of the invention and any modality thereof. As the present invention has already been described in detail, it will be more readily understood by reference to the following examples, which are included herein for purposes of illustration only and which are not intended to be limiting of the invention.

EXAMPLES EXAMPLE 1 Transfection of cells for the activation of endogenous gene expression Method: Construction of pRIG-1 Human DHFR was amplified by PCR from cDNA produced from HT1080 cells by PCR using the DHFR-F1 primers (5 'TCCTTCGAAGCTTGTCATGGTTGGTTCGCTAAACTGCAT 3') (SEQ ID NO: 1) and DHFR-R1 (5 'AAACTTAAGATCGATTAATCATTC-TTCTCATATACTTCAA 3') (SEQ ID NO: 2), and cloned into the T site in pTARGET ™ (Promega) to generate pTARGET: DHFR. The RSV promoter was isolated from PREP9 by digestion with Nhel and Xbal, and inserted into the Nhel site of PTARGET: DHFR to generate pTgT: RSV + DHFR. Oligonucleotides JH169 (5 'ATCCACCATGGCTACAGGTGAGTACTCG 3') (SEQ ID NO: 3) and JH170 (5 ' GATCCGAGTACTCACCTGTAGCCATGGTGGATTTAA 3 ') (SEQ ID NO: 4) were ligated and inserted into the l-Ppo-l and Nhel sites of pTgT: RSV + DHFR to generate pTgT: RSV + DHFR + Exl. A 279 bp region corresponding to nucleotides 230-508 of pBR322 was amplified by PCR using the Tet F1 primers (5 'GGCGAGATCTAGCGCTATATGCGTTGATGCAAT 3') (SEQ ID NO: 5) and Tet F2 (5 'GGCCAGATCTGCTACCTTAAGAGAGCCG-AAACAAGCGCTCATGAGCCCGAA 3') (SEQ ID NO: 6). The amplification products were digested with BglII and cloned into the BamHI site of pTgT: RSV + RSV + DHFR + Exl to generate pRIG-1.

Transfection-Generation of the pRIG-1 gene activation library in HT1080 cells To activate gene expression, an appropriate activation construct is selected from the group of constructions described above. The selected activation construct is then introduced into the cells by any transfection method known in the art. Examples of transfection methods include electroporation, lipofection, calcium phosphate precipitation, DEAE dextran and receptor-mediated endocytosis. After introduction into the cells, the DNA is allowed to integrate into the genome of the host cell by non-homologous recombination. Integration can occur in spontaneous chromosomal breaks or in artificially induced chromosomal breaks.

Method: Transfection of human cells with pRIG-1 2x109 HH1 cells, an HPRT subclone of HT1080 cells, were grown in tissue culture plates of 150 mm up to 90% confluence. The media were removed from the cells and preserved as conditioned media (see below). Cells were removed from the plate by brief incubation with trypsin, added to 10% fetal bovine serum / medium to neutralize trypsin, and pelleted at 1000 rpm in a Jouan centrifuge for 5 minutes. The cells were washed in 1X PBS, counted and transformed again to pellets as indicated above. The cell pellet was resuspended in 2.5 x 107 cells / ml finally in 1X PBS (Gibco BRL, cat # 14200-075). The cells were then exposed to 50 rads of irradiation? from a 137Cs source. PRIG1 was linearized with ßamHI, purified with phenol / chloroform, precipitated with ethanol and resuspended in PBS. The purified and linearized activation construct was added to the cell suspension to produce a final concentration of 40 μg / ml. The DNA / irradiated cell mixture was then mixed, and 400 μl was placed in each 0.4 cm electroporation mixing vessel (Biorad). The mixing vessels were pulsed at 250 Volts, 600 μFarads, 50 Ohms using an electroporation apparatus (Biorad). After the electrical pulse, the cells were incubated at room temperature for 10 minutes, and then placed in 10% MEM / 10% FBS containing penicillin / streptomycin (Gibco / BRL). The cells were then seeded at approximately 7 x 106 cells / 150 mm dish containing 35 ml of MEM alpha / 10% FBS / penstrep (33% conditioned media / 67% fresh media). After an incubation period of 24 hours at 37 ° C, G418 (Gibco / BRL) was added to each plate to a final concentration of 500 μg / ml from a 60 mg / ml supply material. After 4 days of selection, the media were replaced with fresh alpha MEM / 10% FBS / penstrep / 500 μg / ml G418. The cells were then incubated for another 7 to 10 days, and the culture supernatant was tested for the presence of new protein factors or stored at -80 ° C for further analysis. The drug resistant clones were stored in liquid nitrogen for further analysis.

EXAMPLE 2 Use of ionizing irradiation to increase the frequency and randomness of DNA integration Method HH1 cells were harvested at 90% confluence, washed in 1x PBS, and resuspended at a cell concentration of 7.5 x 106 cells / ml in 1X PBS. 15 μg of linearized DNA (pRIG-1) was added to the cells and mixed. 400 μl was added to each mixing vessel for electroporation, and pulsed at 250 Volts, 600 μFarads, 50 Ohms using an electroporation apparatus (Biorad). After the electrical pulse, the cells were incubated at room temperature for 10 minutes, and then placed in 2.5 ml of MEM alpha / 10% FBS / 1X penstrep. 300 μl of the cells of each supply material were irradiated at 0, 50, 500 and 5000 rads immediately before transfection or at 1 hour or 4 hours after transfection. Immediately after irradiation, the cells were seeded in tissue culture plates in complete medium. At 24 hours after sowing, G418 was added to the culture to a final concentration of 500 μg / ml. At 7 days after the selection, the culture medium was replaced with fresh complete medium containing 500 μg / ml of G418. At 10 days after the selection, the medium was removed from the plate, the colonies were stained with Coomassie blue / 90% methanol / 10% acetic acid, and the colonies of more than 50 cells were counted.

EXAMPLE 3 Use of restriction enzymes to generate random, semi-random or targeted breaks in the genome Method HH1 cells were harvested at 90% confluence, washed in 1x PBS, and resuspended at a cell concentration of 7.5 x 106 cells / ml in 1X PBS. To test the integration efficiency, 15 μg of linearized DNA (PGK-βgeo) was added to each aliquot of 400 μl of cells, and mixed. To several aliquots of cells, then the restriction enzymes Xbal,? Foil, Hind \\\, lppo \ (10-500 units) were added to separate the cell / DNA mixture. 400 μl was added to each mixing vessel for electroporation, and pulsed at 250 Volts, 600 μFarads, 50 Ohms using an electroporation apparatus (Biorad). After the electrical pulse, the cells were incubated at room temperature for 10 minutes, and then placed in 2.5 ml of MEM alpha / 10% FBS / IX penstrep. 300 μl of 2.5 ml of the total cells of each supply material were seeded in tissue culture plates in complete media. At 24 hours after sowing, G-418 was added to the culture to a final concentration of 600 μg / ml. At 7 days after the selection, the media was replaced with fresh complete media containing 600 μg / ml of G418. At 10 days after the selection, the media were removed from the plate, the colonies were stained with Coomassie blue / 90% methanol / 10% acetic acid, and the colonies were counted for more than 50 cells.

EXAMPLE 4 Amplification by selection for two amplifiable markers located in the integrated vector After integration of the vector into the genome of a host cell, the genetic locus can be amplified in number of copies by simultaneous or sequential selection for one or more amplifiable markers located in the integrated vector. For example, a vector comprising two amplifiable markers can be integrated into the genome, and the expression of a given gene (i.e., a gene located at the vector's integration site) can be increased by selecting for both amplifiable markers located on the vector . This method greatly facilitates the isolation of clones from cells that have amplified the correct locus (i.e., the locus that contains the integrated vector). Once the vector has been integrated into the genome by non-homologous recombination, individual clones of cells containing the integrated vector can be isolated at a unique position from other cells containing the integrated vector to other positions in the genome. In alternative form, mixed populations of cells can be selected for amplification. Cells containing the integrated vector are then cultured in the presence of a first selective agent that is specific for the first amplifiable marker. This agent selects for cells that have amplified the amplifiable marker in the vector or in the endogenous chromosome. These cells are then selected for the amplification of the second selectable marker by culturing the cells in the presence of a second selective agent that is specific for the second amplifiable marker. The cells that amplified the vector and that flank the genomic DNA will survive this second selective step, while the cells that amplified the first endogenous amplifiable marker or that developed nonspecific resistance will not survive. Additional selections can be carried out similarly when vectors containing more than two (eg, three, four, five or more) amplifiable markers are integrated into the cell genome, by sequential culture of the cells in the presence of selective agents that are specific for the additional amplifiable markers contained in the integrated vector. After selection, the surviving cells are tested to determine the level of expression of a desired gene, and the cells expressing the highest levels are selected for further amplification. Alternatively, groups of cells resistant to both selective agents can be further cultured (if two amplifiable markers are used) or all selective agents (if more than two amplifiable markers are used), without isolation of individual clones. These cells are then expanded and cultured in the presence of higher concentrations of the first selective agent (usually greater than double). The procedure is repeated until the desired level of expression is obtained. Alternatively, cells containing the integrated vector can be selected simultaneously for both amplifiable markers (if two of them are used) or all amplifiable markers (if more than two are used). The simultaneous selection is achieved by incorporating both selection agents (if two markers are used) or all selection agents (if more than two markers are used) in the selection means in which the transfected cells are cultured. The majority of the surviving cells will have amplified the integrated vector. These clones can then be individually selected to identify the cells with the highest expression level, or they can be taken as a group. A higher concentration of each selective agent (usually greater than double) is then applied to the cells. Then, the surviving cells are tested for expression levels. This procedure is repeated until the desired expression levels are obtained. By any selection strategy (ie, simultaneous or sequential selection), the initial concentration of the selective agent is determined independently by titrating the agent from low concentrations without cytotoxicity at high concentrations, which results in the death of the cells in the majority of the cells. In general, a concentration that results in defined colonies (eg, several hundred colonies per 100,000 cells seeded), is chosen as the initial concentration.

EXAMPLE 5 Isolation of cDNA molecules that encode transmembrane proteins The pRIG8RI-CD2 vectors (Fig. 5A-5D; SEQ ID NO: 7), pRIG8R2-CD2 (Fig. 6A-6C; SEQ ID NO: 8 and pRIG8R3-CD2 (Fig. 7A-7C; SEQ ID NO: 9) ) contain the promoter of the CMV immediate early gene operably linked to an exon, followed by an unpaired splice donor site.The exon in the vector codes for a signal peptide linked to the extracellular domain of CD2 (which lacks a codon of arrest in the reading frame.) Each vector codes for CD2 in a different reading frame relative to the splice donor site.To generate a gene library of activated genes, 2 x 107 cells were irradiated with 50 rads from a source of 137Cs and electroporated with 15 μg of linearized pRIG8R-CD2 (SEQ ID NO: 7) .Separately, this was repeated with pRIG8R2-CD2 (SEQ ID NO: 8), and again with pRIG8R3-CD2 SEQ ID NO: 9). After transfection, the three groups of cells were combined and plated in 150 mm plates at 5 x 10 6 transfected cells per plate to generate library # 1. At 24 hours after transfection, library # 1 was placed under 500 μg. / ml of G418 for selection for 14 days. The drug-resistant clones containing the integrated vector in the genome of the host cell were combined, aliquoted and frozen for analysis. Generated library # 2 as described above, except that 3 x 10 7 cells, 3 x 10 7 cells and 1 x 10 7 cells were transfected with pRIG8R2-CD2, pRIG8R2-CD2 and pRIG8R3-CD2, respectively. To isolate the cells containing the activated genes encoding integral membrane proteins, 3 x 10 6 cells from each library were cultured and treated as follows: • The cells were treated with trypsin using 4 ml trypsin-EDTA. • After the cells were released, the trypsin was neutralized by the addition of 8 ml of MEM alpha / 10% FBS. • The cells were washed once with sterile PBS, and collected by centrifugation at 800 x g for 7 minutes. • The cell pellet was resuspended in 2 ml of MEM alpha / 10% FBS. One ml was used for the classification, while the other ml was reseeded in 10% MEM / FBS containing 500 μg / ml of G-418, and it was expanded and conserved. • The cells used for sorting were washed once with sterile alpha MEM / 10% FBS, and collected by centrifugation at 800 x g for 7 minutes. • The supernatant was removed, and the pellet was resuspended in 1 ml of 10% MEM alpha / FBS. 100 μl of these cells were removed for staining with isotype control. • 200 μl of anti-CD2 FITC (Pharmingen, catalog number 30054X), were added to the 900 μl of the cells, while 20 μl of the mouse IgGi isotype control (Pharmingen, catalog number 33814X) were added. 100 μl of the cells. The cells were incubated on ice for 20 minutes. • To the tube containing the cells stained with the anti-human FITC CD2, 5 ml of PBS / 1% FBS was added. To the isotype control, 900 μl of PBS / FBS at 1% was added. Cells were harvested by centrifugation at 600 x g for 6 minutes. • The supernatant was removed from the tubes. Cells that had been stained with the isotype control were resuspended in 500 μl of 10% MEM alpha / FBS, and the cells that had been stained with anti-CD2 FITC were resuspended in 1.5 ml of MEM alpha / FBS a 10% The cells were sorted through five sequential classes in a FACS Vantage flow cytometer (Becton Dickinson Immunocytometry Systems, Mountain View, CA). In each class, the indicated percentage of the total cells, representing the most strongly fluorescent cells (see below) was collected, expanded and reclassified. The HT1080 cells were classified as negative control. The following populations were classified and collected in each class: The cells from each of the final classes of each library were expanded and stored in liquid nitrogen.

Isolation of activated cells from cells classified by FACS Once the cells were classified as described above, the endogenous genes activated from the sorted cells were isolated by PCR-based cloning. However, one skilled in the art will appreciate that any method known in the art of gene cloning can be used in an equivalent manner to isolate activated genes from cells classified by FACS. The genes were isolated by the following protocol: 1) Using the PoIyATract 1000 messenger RNA isolation kit (Promega), messenger RNA was isolated from 3x107 CD2 + cells (five cycles sorted by FACS, as described above) from the libraries number 1 and 2. 2) After the isolation of the messenger RNA, the concentration of the messenger RNA was determined by diluting 0.5 μl of isolated messenger RNA, in 99.5 μl of water, and measuring the OD260. 25 μg of messenger RNA was recovered from the CD2 + cells. 3) First-strand cDNA synthesis was then carried out in the following manner: a) While the PCR machine was maintained at 4 ° C; the reaction mixtures of the first chain were adjusted by the sequential addition of the following components: 41 μl of ddH20 treated with DEPC 4 μl of dNTP, each at 10 mM 8 μl of MDTT at 0.1 M 16 μl of 5x pH regulator for first MMLV chain (Gibco-BRL) 5 μl (10 pmol / μl) of the consensus polyadenylation site initiator GD.R1 (SEQ ID NO: 10) * 1 μl of RNAs (Promega) 3 μl (1.25 μg / μl) of Messenger RNA * Note: GD.R1, 5 'TTTTTTTTTTTTCGTCAGCGGCCGCATCNNNNTTT-ATT 3' (SEQ ID NO: 10), is an initiator for the "Discovery of genes" for the synthesis of first strand cDNA messenger RNA; this initiator is designed to bind to the AATAAA polyadenylation signal and the poly-A region towards the 3 'end. This initiator will introduce a site? / Oíl in the first string. Once the samples were obtained, they were incubated as follows: b) 70 ° C for 1 minute c) maintenance at 42 ° C. 2 μl of 400 U / μl SuperScript II (Gibco-BRL, Rockville, MD) was then added to each sample to give a final total volume of 82 μl. After approximately three minutes, the samples were incubated as follows: d) 37 ° C for 30 min. e) 94 ° C for 2 min. f) 4 ° C for 5 min. Then 2 μl of 20 U / μl of RNace-IT (Stratagene) was added to each sample, and the samples were incubated at 37 ° C for 10 minutes. 4) After the synthesis of the first strand, cDNA was purified using a PCR cleaning kit (Qiagen) in the following manner: a) 80 μl of the reaction of the first strand was transferred to a 1.7 ml siliconized Eppendorf tube , and adding 400 μl of PB. b) The samples were then transferred to a PCR cleaning column, and centrifuged for two minutes at 14,000 RPM. c) The columns were then disassembled, decanted completely, 750 μl of PE was added to the pellets, and the tubes were centrifuged for two minutes at 14,000 RPM. d) The columns were disassembled and decanted completely, and the tubes were then centrifuged for two minutes at 14,000 RPM to dry the resin. e) The cDNA was then eluted using 50 μl of EB, through a transfer column, to a new siliconized Eppendorf tube, which was then centrifuged for two minutes at 14,000 RPM. 5) The synthesis of the cDNA of the second chain was carried out in the following manner: a) The reaction mixtures of the second chain were adjusted to room temperature, by the sequential addition of the following components: ddH20 55 μl 10 x regulator of pH for PCR 10 μl MgCl2 at 50 mM 5 μl dNTPs at 10 mM 2 μl 25 pmol / μl of RIG. 751 -Bio * 4 μl 25 pmoles / μl of GD. R2 ** 4 μl Product of the first chain 20 μl * Note: RIG.F751-BÍO, 5 'Biotin-CAGATCACTAGAAGCTTTATTGCGG 3' (SEQ ID NO: 11), binds to the cap site of the expressed transcript of pRIG vectors. ** Note: GD.R2, 5 'TTTTCGTCAGCGGCCGCATC 3' (SEQ ID NO: 12), is an initiator used to PCR amplify cDNA molecules generated using the primer GD.R1 (SEQ ID NO: 10). GD.R2 is a subsequence of GD.R1 with a mating sequence with the degenerate bases preceding the signal sequence poIyA. b) Start the synthesis of the second chain: 94 ° C for 1 min; add 1 μl of Taq (5U / μl, Gibco-BRL); add 1 μl of Vent DNA polymerase (0.1 U / μl, New England Biolabs) c) Incubate at 63 ° C for 2 minutes d) Incubate at 72 ° C for 3 minutes e) Repeat step four times b) f) Incubate 72 ° C for 6 minutes g) Incubate at 4 ° C (keep at this temperature) h) End of the procedure. 6) 200 μl of 1 mg / ml of streptavidin-paramagnetic particles (SA-PMP) was then prepared by washing three times with STE 7) The products of the reaction of the second chain were added directly to SA-PMPs, and incubated room temperature for 30 minutes 8) After the binding, SA-PMPs were collected by the use of the magnet, and the material was fully recovered 9) The spheres were washed three times with 500 μl of STE 10) The spheres were resuspended in 50 μl of STE, and collected at the bottom of the tube using the magnet. The supernatant of STE was then carefully pipetted. 11) The beads were resuspended in 50 μl of ddH20, and placed in a water bath at 100 ° C for two minutes, to release the purified cDNA from PMPs. 12) Purified cDNA was recovered by collecting PMPs in the magnet, and carefully removing the supernatant containing the cDNA. 13) The purified products were transferred to a clean tube, and centrifuged at 14,000 RPM for two minutes to remove all residual PMPs. 14) A PCR reaction was then carried out to specifically amplify RIG-activated cDNA molecules from the as follows: a) The PCR reaction mixtures were adjusted to room temperature, by sequential addition of the following components: H20 59 μl 10 x pH regulator for 10 μl PCR MgCI2 at 50 mM 5 μl DNTPs at 10 mM 2 μl 25 pmol / μl of RIG. 2 μl F781 * 25 pmol / μl of GD.R2 2 μl Product of the second 20 μl chain * Note RIG.F781, 5 'ACTCATAGGCCATAGAGGCCTATCACAG- TTAAATTGCTAACGCAG 3' (SEQ ID NO: 13), binds to the 3 'end of GD.F1 GD.F3, GD.F5-Bio and RIG.F751-Bio, and adds a Sfi \ site for the 'cloning of cDNA molecules. This initiator is used in the amplification nested PCR of second-strand cDNA molecules specific for exon 1 of RIG. b) The thermal cycler is operated: 94 ° C for 3 minutes; add 1 μl of Taq (5U / μl, Gibco-BRL); add 1 μl of Vent DNA polymerase to 0.1 U / μl (New England Biolabs) PCR was then carried out by 10 cycles of steps c) to e): c) 94 ° C for 30 sec. d) 60 ° C for 40 sec. e) 72 ° C for 3 min. The PCR was then concluded carrying out the following steps: f) 94 ° C for 30 sec. g) 60 ° C for 40 sec. h) 72 ° C for 3 min. i) 72 ° C + each cycle of 20 sec. for 10 cycles j) 72 ° C for 5 min. k) maintenance at 4 ° C. 15) After elution of the library material with 50 μl of EB, the samples were digested by adding 10 μl of buffer pH 2 NEB, 40 μl of dH2Oμl of Sf / 1, and digesting for 1 hour at 50 ° C, to cut the 5 'end of the cDNA at the Sfil site encoded by the forward primer (RIG.F781; SEQ ID NO: 13). 16) After digestion of Sfil, 5 μl of 1 M NaCl and 2 μl of Notl were added to each sample, and the samples were digested for 1 hour at 37 ° C, to cut the 3 'end of the cDNA in the Notl site encoded by the first chain primer (GD.R1; SEQ ID NO: 10). 17) The digested cDNA was then separated on a 1% low melting point agarose gel. Gel molecules were separated from the gel CDNAs that varied in size from 1.2Kb to 8Kb. 18) cDNA was recovered from the separated agarose gel using Qiaex II gel extraction (Qiagen). 2 μl of cDNA (approximately 30 mg) were ligated to 7 μl (35 ng) of pBS-HSB (linearized with Sfil / Notl) in a total volume of 10 μl of 1X pH regulator for T4 ligase (NEB), using 400 units of T4 DNA ligase (NEB). 19) 0.5 μl of the reaction mixture for ligation of step (18), were transformed into DH10B of E. coli. 20) 103 colonies / 0.5 μl of bound DNA was recovered. 21) These colonies were selected for exons using primers M13F20 and JH182 (specific for exon 1 of RIG) by PCR in volumes of 12.5 μl, as follows: a) 100 μl of LB (with selective antibiotic) were supplied in the Appropriate number of 96-well plates. b) The individual colonies were selected and inoculated in the individual wells of the 96-well plate, and the plate was placed in an incubator at 37 ° C for 2 to 3 hours without agitation. c) A "master mix" was prepared for PCR reaction on ice, as follows: d) 10 μl of the master mix were supplied in each well of the PCR reaction plate e) 2.5 μl of each 100 μl of the E. coli culture were transferred to the corresponding wells of the PCR reaction plate. f) PCR was carried out, using typical conditions of the PCR cycle of: (i) 94 ° C / 2min. (bacterial lysis and denaturation of plasmid); (ii) 30 cycles of denaturation at 92 ° C for 15 seconds; primer binding at 60 ° C for 20 seconds; and extension of the initiator at 72 ° C for 40 seconds; (iii) final extension at 72 ° C for 5 minutes; (iv) maintenance at 4 ° C. g) Bromophenol blue was then added to the PCR reaction; the samples were mixed and centrifuged, and then the entire reaction mixture was loaded onto an agarose gel. 23) Of 200 selected clones, 78% were positive for the exon of the vector. 96 of these clones were developed as mini-preparations, and purified using a Qiagen 96-well turbo preparation, following the mini-preparations manual Qiagen (April 1997). 24) Many duplicate clones were eliminated through the simultaneous digestion of 2 μl of DNA with Notl, Bam Hl, Xhol, Xbal, Hindlll and EcoRl in buffer pH 3 NEB, in a total volume of 22 μl, followed by electrophoresis on a 1% agarose gel.

Results: Two different cDNA libraries were selected using this protocol. In the first library (TMT # 1), eight of the isolated activated genes were sequenced. Of these eight genes, four genes encoded for known integral membrane proteins, and six were novel genes. In the second library (TMT # 2), 11 isolated activated genes were sequenced. Of these 11 genes, one gene encoded for a well-known integral membrane protein, one gene encoded for a partially sequenced gene homologous to an integral membrane protein, and nine were novel genes. In all cases where the isolated gene corresponded to a known characterized gene, said gene was an integral membrane protein. Below are examples of significant alignments (obtained from the Gene Bank) for genes isolated from each library: Significant alignments of TMT # 1: 179761 | gb | M76559 | HUMCACNLB: Complete CDs of messenger RNA of the calcium channel alpha-2b subunit, voltage dependent and sensitive to human neuronal DHP. Length = 3600 > gi | 3183974 | emb | Y10183 | HSMEMD: messenger RNA for MEMD protein of H. sapiens. Length = 4235 Significant TMT # 2 alignments: > gi | 476590 | gb | U06715 | HSU06715: human cytochrome B561, HCYTO B561, messenger RNA, partial CDs. Length = 2463 > gi | 2184843 | gb | AA459959 | AA459959 ZX66C01.S1: Nb2HF8 9w total of Soares fetus; clone 796414 of Homo sapiens cDNA 3 'similar to the precursor of the gb interferon alpha receptor: J03171 (human); length = 431 Having now fully described the present invention in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious to the person skilled in the art that it can be carried out by modifying or changing the invention within a broad and equivalent scale of conditions, formulations and other parameters, without affecting the scope of the invention or any specific modality thereof, and that said modifications or changes are intended to be contemplated within the scope of the appended claims. All publications, patents and patent applications mentioned in this description of the invention are indicative of the level of aptitude of those skilled in the art to which this invention pertains, and are incorporated herein by reference to the same extent as if each publication, patent or individual patent application was indicated in a specific and individual form to be incorporated herein by reference.

LIST OF SEQUENCES < 110 > Harrington, John J. Athersys, Inc. < 120 > Expression of endogenous genes by non-homologous recombination of a vector construct with cellular DNA < 130 > 1522.003PC01 < 140 > To be assigned < 141 > 1998-09-25 < 150 > To be assigned < 151 > 1998-09-24 < 150 08/941, 223 < 151 > 1997-09-26 < 160 > 13 < 170 > Patentln Ver. 2.0 < 210 > 1 < 211 > 39 < 212 > DNA < 213 > Homo sapiens < 400 > 1 tccttcgaag cttgtcatgg ttggttcgct aaactgcat 39 < 210 > 2 < 211 > 40 < 212 > DNA < 213 > Homo sapiens < 400 > 2 aaacttaaga tcgattaatc attcttctca tatacttcaa 40e. < 210 > 3 < 211 > 28 < 212 > DNA < 213 > Homo sapiens < 400 > 3 atccaccatg gctacaggtg agtactcg 28 < 210 > 4 < 211 > 36 < 212 > DNA < 213 > Homo sapiens < 400 > 4 gatccgagta ctcacctgta gccatggtgg atttaa 36 < 210 > 5 < 211 > 33 < 212 > DNA < 213 > Homo sapiens < 400 > 5 ggcgagatct agcgctatat gcgttgatgc aat 33 < 210 > 6 < 211 > 51 < 212 > DNA < 213 > Homo sapiens < 400 > 6 ggccagatct gctaccttaa gagagccgaa acaagcgctc atgagcccga a 51 < 210 > 7 < 211 > 6084 < 212 > DNA < 213 > Homo sapiens < 400 > 7 agatcttcaa tattggccat tagccatatt attcattggt tatatagcat aaatcaatat 60 tggctattgg ccattgcata cgttgtatct atatcataat atgtacattt atattggctc 120 atgtccaata tgaccgccat gttggcattg attattgact agttattaat agtaatcaat 180 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 240 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 300 acgccaatag tcccatagta ttgacgtcaa ggactttcca atttacggta tgggtggagt 360 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtccgcccc ctattgacgt 420 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttac gggactttcc 480 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca 540 gtacaccaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat 600 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa 660 caactgcgat cgcccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc 720 tatataagca gagctcgttt agtgaaccgt cagatcacta gaagctttat tgcggtagtt 780 tatcacagtt aaattgctaa cgcagtcagt gcttctgaca caacagtctc gaacttaagc 840 tgcagtgact ctcttaat ta actccaccag tctcacttca gttccttttg cctccaccag 900 tctcacttca gttccttttg catgaagagc tcagaatcaa aagaggaaac caacccctaa 960 gatgagcttt ccatgtaaat ttgtagccag cttccttctg attttcaatg tttcttccaa 1020 aggtgcagtc tccaaagaga ttacgaatgc cttggaaacc tggggtgcct tgggtcagga 1080 catcaacttg gacattccta gttttcaaat gagtgatgat attgacgata taaaatggga 1140 gacaagaaaa aaaaacttca agattgcaca attcagaaaa ctttcaagga gagaaagaga 1200 aaaagataca tataagctat ttaaaaatgg aactctgaaa attaagcatc tgaagaccga 1260 tgatcaggat atctacaagg tatcaatata tgatacaaaa ggaaaaaatg tgttggaaaa 1320 aatatttgat ttgaagattc aagagagggt ctcaaaacca aagatctcct ggacttgtat 1380 caacacaacc ctgacctgtg aggtaatgaa tggaactgac acctgtatca cccgaattaa 1440 agatgggaaa catctaaaac tttctcagag ggtcatcaca cacaagtgga ccaccagcct 1500 ttcaagtgca gagtgcaaaa cagcagggaa caaagtcagc aaggaatcca gtgtcgagcc 1560 tgtcagctgt ccagagaaag ggatccaggt gagtagggcc cgatccttct agagtcgagc 1620 tctcttaagg tagcaaggtt acaagacagg tttaaggaga ccaatagaaa ctgggcttgt 1680 cgagacagag aagactcttg CGTT tctgat aggcacctat tggtcttacg cggccgcgaa 1740 ttccaagctt gagtattcta tcgtgtcacc taaataactt ggcgtaatca tggtcatatc 1800 tgtttcctgt gtgaaattgt caattccaca tatccgctca gccggaagca caacatacga 1860 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgcg 1920 atgcttccat tttgtgaggg ttaatgcttc gagaagacat gataagatac attgatgagt 1980 ttggacaaac atgcagtgaa cacaacaaga aaaaatgctt tatttgtgaa atttgtgatg 2040 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca 2100 ttcattttat gtttcaggtt cagggggaga tgtgggaggt tttttaaagc aagtaaaacc 2160 tctacaaatg tggtaaaatc cgataaggat cgattccgga gcctgaatgg cgaatggacg 2220 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcacg tgaccgctac 2280 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt 2340 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc 2400 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc 2460 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact 2520 actggaacaa cttgttccaa cactcaacc c tatctcggtc tattcttttg atttataagg 2580 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc 2640 gaattttaac aaaatattaa cgcttacaat ttcgcctgtg taccttctga ggcggaaaga 2700 accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca 2760 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct 2820 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc 2880 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg 2940 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc 3000 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctt gattcttctg 3060 acacaacagt ctcgaactta aggctagagc caccatgatt gaacaagatg gattgcacgc 3120 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat 3180 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt 3240 caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg 3300 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag 3360 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc 3420 gtatccatca tgccgagaaa tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc 3480 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag ctcggatgga cgagcacgta 3540 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga 3600 actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg 3660 ttgccgaata cgatgcctgc tcatggtgga aaatggccgc ttttctggat tcatcgactg 3720 t ggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc 3780 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc 3840 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg 3900 tgaccgacca gggttcgaaa agcgacgccc aacctgccat cacgatggcc gcaataaaat 3960 atctttattt tcattacatc tgtgtgttgg ttttttgtgt gaagatccgc gtatggtgca 4020 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac 4080 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc caagctgtga cgcttacaga 4140 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac 4200 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt 4260 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct 4320 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat 4380 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg 4440 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 4500 aagatcagtt gggtgcacga tcgaactgga gtgggttaca tctcaacagc ggtaagatcc 4560 ttgaga GTTT tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat 4620 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact 4680 attctcagaa tgacttggtt gagtactcac aaagcatctt cagtcacaga acggatggca 4740 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 4800 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg 4860 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 4920 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg 4980 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg 5040 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 5100 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc 5160 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga 5220 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat 5280 gattgattta atatacttta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 5340 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 5400 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 5460 aaaaaaacca gcttgcaaac ccgctaccag cggtggtttg tttgccggat caagagctac 5520 tccgaaggta caactctttt actggcttca gcagagcgca gataccaaat actgtccttc 5580 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 5640 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 5700 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 5760 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 5820 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 5880 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 5940 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 6000 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 6060 ggccttttgc tcacatggct CGAC 6084 < 210 > 8 < 211 > 6085 < 212 > DNA < 213 > Homo sapiens < 400 > 8 agatcttcaa tattggccat tagccatatt attcattggt tatatagcat aaatcaatat 60 tggctattgg ccattgcata cgttgtatct atatcataat atgtacattt atattggctc 120 atgtccaata tgaccgccat gttggcattg attattgact agttattaat agtaatcaat 180 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 240 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 300 tcccatagta acgccaatag ggactttcca ttgacgtcaa atttacggta tgggtggagt 360 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtccgcccc ctattgacgt 420 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttac gggactttcc 480 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca 540 gtacaccaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat 600 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa 660 caactgcgat cgcccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc 720 tatataagca gagctcgttt agtgaaccgt cagatcacta gaagctttat tgcggtagtt 780 tatcacagtt aaattgctaa cgcagtcagt gcttctgaca caacagtctc gaacttaagc 840 tgcagtgact ctcttaat ta actccaccag tctcacttca gttccttttg cctccaccag 900 tctcacttca gttccttttg catgaagagc tcagaatcaa aagaggaaac caacccctaa 960 gatgagcttt ccatgtaaat ttgtagccag cttccttctg attttcaatg tttcttccaa 1020 aggtgcagtc tccaaagaga ttacgaatgc cttggaaacc tggggtgcct tgggtcagga 1080 catcaacttg gacattccta gttttcaaat gagtgatgat attgacgata taaaatggga 1140 gacaagaaaa aaaaacttca agattgcaca attcagaaaa ctttcaagga gagaaagaga 1200 aaaagataca tataagctat ttaaaaatgg aactctgaaa attaagcatc tgaagaccga 1260 tgatcaggat atctacaagg tatcaatata tgatacaaaa ggaaaaaatg tgttggaaaa 1320 aatatttgat ttgaagattc aagagagggt ctcaaaacca aagatctcct ggacttgtat 1380 caacacaacc ctgacctgtg aggtaatgaa tggaactgac acctgtatca cccgaattaa 1440 agatgggaaa catctaaaac tttctcagag ggtcatcaca cacaagtgga ccaccagcct 1500 ttcaagtgca gagtgcaaaa cagcagggaa caaagtcagc aaggaatcca gtgtcgagcc 1560 tgtcagctgt ccagagaaag ggatcccagg tgagtagggc ccgatccttc tagagtcgag 1620 ctctcttaag gtagcaaggt tacaagacag gtttaaggag accaatagaa actgggcttg 1680 tcgagacaga gaagactctt gcgt ttctga ttggtcttac taggcaccta gcggccgcga 1740 attccaagct tgagtattct atcgtgtcac ctaaataact tggcgtaatc atggtcatat 1800 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc 1860 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc 1920 gatgcttcca ttttgtgagg gttaatgctt cgagaagaca tgataagata cattgatgag 1980 tttggacaaa ccacaacaag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat 2040 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc 2100 attcatttta tgtttcaggt tcagggggag atgtgggagg ttttttaaag caagtaaaac 2160 ctctacaaat gtggtaaaat ccgataagga tcgattccgg agcctgaatg gcgaatggac 2220 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcac gtgaccgcta 2280 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt 2340 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg 2400 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat 2460 gacggttttt cgccctgata cgccctttga cgttggagtc cacgttcttt aatagtggac 2520 aactggaaca tcttgttcca acactcaacc ctatctcggt ctattctttt gatttataag 2580 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg 2640 caaaatatta cgaattttaa acgcttacaa tttcgcctgt gtaccttctg aggcggaaag 2700 aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc 2760 aaagcatgca agaagtatgc tcagcaacca tctcaattag ggtgtggaaa gtccccaggc 2820 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg 2880 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat 2940 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc 3000 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct tgattcttct 3060 gacacaacag tctcgaactt aaggctagag ccaccatgat tgaacaagat ggattgcacg 3120 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa 3180 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg 3240 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt 3300 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa 3360 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc 3420 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg 3480 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg 3540 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg 3600 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg 3660 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact 3720 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg 3780 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc 3840 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct 3900 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgatggc cgcaataaaa 3960 tatctttatt ttcattacat ctgtgtgttg gttttttgtg tgaagatccg cgtatggtgc 4020 actctcagta tgatgccgca caatctgctc tagttaagcc agccccgaca cccgccaaca 4080 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg 4140 accgtctccg ggagctgcat gtgtc agagg ttttcaccgt catcaccgaa acgcgcgaga 4200 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct 4260 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 4320 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 4380 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 4440 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 4500 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 4560 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 4620 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac 4680 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 4740 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 4800 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 4860 ctcgccttga gatcatgtaa tcgttgggaa ccggagctga atgaagccat accaaacgac 4920 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc 4980 ctctagcttc gaactactta ccggcaacaa ttaatagact ggatggaggc ggataaagtt 5040 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 5100 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 5160 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 5220 atcgctgaga taggtgcctc actgattaag cattggtaac agtttactca tgtcagacca 5280 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 5340 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 5400 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 5460 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt tcaagagcta gtttgccgga 5520 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt 5580 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 5640 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 5700 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 5760 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 5820 ctatgagaaa gcgccacgct tcccgaaggg agaaa GGCGG acaggtatcc ggtaagcggc 5880 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 5940 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 6000 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 6060 tggccttttg ctcacatggc tcgac 6085 < 210 > 9 < 211 > 6086 < 212 > DNA < 213 > Homo sapiens < 400 > 9 agatcttcaa tattggccat tagccatatt attcattggt tatatagcat aaatcaatat 60 tggctattgg ccattgcata cgttgtatct atatcataat atgtacattt atattggctc 120 atgtccaata tgaccgccat gttggcattg attattgact agttattaat agtaatcaat 180 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 240 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 300 acgccaatag tcccatagta ttgacgtcaa ggactttcca atttacggta tgggtggagt 360 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtccgcccc ctattgacgt 420 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttac gggactttcc 480 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca 540 gtacaccaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat 600 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa 660 caactgcgat cgcccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc 720 tatataagca gagctcgttt agtgaaccgt cagatcacta gaagctttat tgcggtagtt 780 tatcacagtt aaattgctaa cgcagtcagt gcttctgaca caacagtctc gaacttaagc 840 tgcagtgact ctcttaa tta actccaccag tctcacttca gttccttttg cctccaccag 900 tctcacttca gttccttttg catgaagagc tcagaatcaa aagaggaaac caacccctaa 960 gatgagcttt ccatgtaaat ttgtagccag cttccttctg attttcaatg tttcttccaa 1020 aggtgcagtc tccaaagaga ttacgaatgc cttggaaacc tggggtgcct tgggtcagga 1080 catcaacttg gacattccta gttttcaaat gagtgatgat attgacgata taaaatggga 1140 gacaagaaaa aaaaacttca agattgcaca attcagaaaa gagaaagaga ctttcaagga 1200 aaaagataca tataagctat ttaaaaatgg aactctgaaa attaagcatc tgaagaccga 1260 tgatcaggat atctacaagg tatcaatata tgatacaaaa ggaaaaaatg tgttggaaaa 1320 aatatttgat ttgaagattc aagagagggt ctcaaaacca aagatctcct ggacttgtat 1380 caacacaacc ctgacctgtg aggtaatgaa tggaactgac acctgtatca cccgaattaa 1440 agatgggaaa catctaaaac tttctcagag ggtcatcaca cacaagtgga ccaccagcct 1500 ttcaagtgca gagtgcaaaa caaagtcagc cagcagggaa aaggaatcca gtgtcgagcc 1560 tgtcagctgt ccagagaaag ggatccacag gtgagtaggg cccgatcctt ctagagtcga 1620 ggtagcaagg gctctcttaa ggtttaagga ttacaagaca gaccaataga aactgggctt 1680 gtcgagacag agaagactct tgc gtttctg ataggcacct attggtctta cgcggccgcg 1740 aattccaagc ttgagtattc tatcgtgtca cctaaataac ttggcgtaat catggtcata 1800 tctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag 1860 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg 1920 cgatgcttcc attttgtgag ggttaatgct tcgagaagac atgataagat acattgatga 1980 accacaacaa gtttggacaa gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga 2040 ttatttgtaa tgctattgct ccattataag ctgcaataaa caagttaaca acaacaattg 2100 cattcatttt atgtttcagg ttcaggggga gatgtgggag gttttttaaa gcaagtaaaa 2160 tgtggtaaaa cctctacaaa tccgataagg atcgattccg gagcctgaat ggcgaatgga 2220 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca cgtgaccgct 2280 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg 2340 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt 2400 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca 2460 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga 2520 ctcttgttcc aaactggaac aacactcaa c cctatctcgg tctattcttt tgatttataa 2580 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac 2640 gcgaatttta acaaaatatt aacgcttaca atttcgcctg tgtaccttct gaggcggaaa 2700 gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg 2760 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg 2820 ggcagaagta ctccccagca tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc 2880 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca 2940 atttttttta tggctgacta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt 3000 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc ttgattcttc 3060 gtctcgaact tgacacaaca gccaccatga taaggctaga ttgaacaaga tggattgcac 3120 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca 3180 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt 3240 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg 3300 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga 3360 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct 3420 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg 3480 cattcgacca gctacctgcc ccaagcgaaa catcgcatcg agcgagcacg tactcggatg 3540 ttgtcgatca gaagccggtc ggatgatctg gacgaagagc atcaggggct cgcgccagcc 3600 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat 3660 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac 3720 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt 3780 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct 3840 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc 3900 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgatgg ccgcaataaa 3960 atatctttat tttcattaca tctgtgtgtt ggttttttgt gtgaagatcc gcgtatggtg 4020 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac 4080 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt 4140 gggagctgca gaccgtctcc tgtgtc agag gttttcaccg tcatcaccga aacgcgcgag 4200 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc 4260 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt 4320 ctaaatacat tcaaatatgt atccgctcat gagacaataa tgcttcaata ccctgataaa 4380 aggaagagta atattgaaaa tgagtattca acatttccgt gtcgccctta ttcccttttt 4440 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc 4500 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat 4560 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct 4620 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca 4680 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg 4740 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa 4800 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg 4860 ggatcatgta actcgccttg atcgttggga accggagctg taccaaacga aatgaagcca 4920 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg 4980 actctagctt cgaactactt cccggcaaca attaatagac tggatggagg cggataaagt 5040 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg 5100 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc 5160 gttatctaca ccgtatcgta cgacggggag tcaggcaact atggatgaac gaaatagaca 5220 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc 5280 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat 5340 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc 5400 gaaaagatca agaccccgta aaggatcttc ttgagatcct ttttttctgc gcgtaatctg 5460 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct 5520 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct 5580 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct 5640 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg 5700 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc 5760 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga 5820 gctatgagaa agcgccacgc ttcccgaagg Gagaa aggcg gacaggtatc cggtaagcgg 5880 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta 5940 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 6000 ctatggaaaa ggggcggagc acgccagcaa cgcggccttt ttacggttcc tggccttttg 6060 ctggcctttt gctcacatgg ctcgac 6086 < 210 > 10 < 211 > 38 < 212 > DNA < 213 > Homo sapiens < 400 > 10 tttttttttt ttcgtcagcg gccgcatcnn nntttatt 38 < 210 > 11 < 211 > 25 < 212 > DNA < 213 > Homo sapiens < 400 > 11 cagatcacta gaagctttat tgcgg 25 < 210 > 12 < 211 > 20 < 212 > DNA < 213 > Homo sapiens < 400 > 12 ttttcgtcag cggccgcatc 20 < 210 > 13 < 211 > 45 < 212 > DNA < 213 > Homo sapiens < 400 > 13 actcataggc catagaggcc tatcacagtt aaattgctaa cgcag 45

Claims (95)

1. NOVELTY OF THE INVENTION CLAIMS 1. - A vector construct consisting essentially of a transcriptional regulatory sequence operably linked to an unpaired sequence of splice donor, and one or more amplifiable markers.
2. 2. A vector construct consisting essentially of a transcriptional regulatory sequence operably linked to a start codon of translation, a signal signal of secretion, and an unpaired site of splice donor.
3. 3. A vector construct consisting essentially of a transcriptional regulatory sequence operably linked to a translation initiation codon, an epitope tag and an unpaired splice donor site.
4. 4. A vector construct comprising a transcriptional regulatory sequence operably linked to a translation initiation codon, a secretion signal sequence, an epitope tag, and an unpaired splice donor site.
5. 5. A vector construct comprising a transcriptional regulatory sequence operably linked to a translation initiation codon, a secretion signal sequence, an epitope tag, a sequence specific protease site, and an unpaired site of splice donor.
6. 6. The vector construction according to any of claims 2 to 5, further characterized in that said construction further comprises an internal site for entry of the ribosome, to produce a polycistronic message.
7. 7. The vector construction according to any of claims 2 to 5, further characterized in that said construction further comprises one or more amplifiable markers.
8. 8. The vector construction according to claim 6, further characterized in that said construction further comprises one or more amplifiable markers.
9. 9. The vector construction according to any of claims 1 to 5, further characterized in that said transcription regulatory sequence is a promoter.
10. 10. The vector construction according to claim 9, further characterized in that said promoter is a viral promoter.
11. 11. The vector construction according to claim 10, further characterized in that said viral promoter is an immediate early promoter of cytomegalovirus.
12. 12. The vector construction according to claim 9, further characterized in that said promoter is a non-viral promoter.
13. 13. The vector construction according to claim 9, further characterized in that said promoter is an inducible promoter.
14. 14. The vector construction according to any of claims 1 to 5, further characterized in that said transcription regulatory sequence is an enhancer.
15. 15. The vector construction according to claim 14, further characterized in that said enhancer is a viral enhancer.
16. 16. The vector construction according to claim 15, further characterized in that said viral enhancer is an immediate early enhancer of cytomegalovirus.
17. 17. The vector construction according to claim 14, further characterized in that said enhancer is a non-viral enhancer.
18. 18. A cell containing the vector construct according to any of claims 1 to 5.
19. 19. The cell according to claim 18, further characterized in that said vector construction has been integrated into the cellular genome.
20. 20. The cell according to claim 19, further characterized in that an endogenous gene is overexpressed in said cell by upregulating the gene by said transcription regulatory sequence on said vector construct.
21. 21. The cell according to claim 18, further characterized in that said cell is an isolated cell.
22. 22. A method for obtaining a recombinant cell, characterized in that it comprises introducing the construction according to any of claims 1 to 5 into a cell.
23. 23. A method for overexpressing an endogenous gene in a cell, characterized in that it comprises: a) introducing the construction according to any of claims 1 to 5 into a cell; b) allowing said construction to be integrated into the genome of said cell by non-homologous recombination; and c) allowing overexpression of said endogenous gene in said cell.
24. 24. The method according to claim 23, further characterized in that said overexpression is achieved in vitro.
25. 25. The method according to claim 23, further characterized in that said overexpression is achieved in vivo.
26. 26. A method for producing an expression product isolated from an endogenous cellular gene, characterized in that it comprises: a) overexpressing an endogenous gene in a cell according to the method according to claim 23, wherein an expression product of said endogenous gene is produced by said cell; and b) isolating said expression product from said cell.
27. 27. A cell library comprising a collection of cells transformed with the construction according to any of claims 1 to 5, characterized in that said construction is integrated into the genomes of said cells by non-homologous recombination.
28. 28. A method for obtaining an overexpressed gene product from a cell library comprising selecting the library according to claim 27, for the expression of said gene product, selecting from said library a cell overexpressing said gene product. , and obtain said gene product from said selected cell.
29. 29. A method for producing an expression product isolated from an endogenous cellular gene, characterized in that it comprises: a) introducing a vector comprising a transcription regulatory sequence in a cell; b) allowing said vector to be integrated into the genome of said cell by non-homologous recombination; c) allowing overexpression of an endogenous gene in said cell by upregulating said gene by said transcription regulatory sequence; d) selecting said cell for overexpression of said endogenous gene; e) culturing said cell under conditions that favor the production of the expression product of said endogenous gene by said cell; and f) isolating said expression product.
30. 30. A method for producing an expression product of an endogenous cellular gene, characterized in that it comprises: a) introducing a vector comprising a regulatory sequence of the non-retroviral transcription in a cell, b) allowing said vector to be integrated into the cell. genome of said cell by non-homologous recombination, c) allowing the overexpression of an endogenous gene in said cell by upregulating said gene by said transcription regulatory sequence, d) selecting said cell for overexpression of said endogenous gene, and e) cultivating said cell under conditions that favor the production of the expression product of said endogenous gene by said cell. 31.- A method for producing an expression product of an endogenous cellular gene, characterized in that it comprises: a) introducing a vector comprising a transcription regulatory sequence operably linked to a secretion signal sequence in a cell, b) allowing said vector is integrated into the genome of said cell by non-homologous recombination, c) allowing overexpression of an endogenous gene in said cell by upregulating said gene by said transcription regulatory sequence, d) selecting said cell for overexpression of said gene. said endogenous gene, and e) culturing said cell under conditions that favor the production of the expression product of said endogenous gene by said cell. 32. A method for producing an expression product of an endogenous cellular gene, characterized in that it comprises: a) introducing a vector comprising a regulatory sequence of the non-retroviral transcription operably linked to a signal sequence of secretion in a cell, b) allowing said vector to be integrated into the genome of said cell by non-homologous recombination, c) allowing the overexpression of an endogenous gene in said cell by up-regulating said gene by said transcription regulatory sequence, d) selecting said cell for the overexpression of said endogenous gene, and e) culturing said cell under conditions that favor the production of the expression product of said endogenous gene by said cell. 33.- A method for producing an expression product of an endogenous cellular gene, characterized in that it comprises: a) introducing a vector comprising a transcriptional regulatory sequence operably linked to an unpaired sequence of splice donor in a cell, b ) allowing said vector to be integrated into the genome of said cell by non-homologous recombination, c) allowing the overexpression of an endogenous gene in said cell by upregulating said gene by said transcription regulatory sequence, d) selecting said cell for overexpression of said endogenous gene, and e) culturing said cell under conditions that favor the production of the expression product of said endogenous gene by said cell. 34. The method according to any of claims 30 to 33, further characterized in that it comprises isolating said expression product. 35.- A method for overexpressing an endogenous gene in a cell in vivo, characterized in that it comprises: a) introducing a vector comprising a regulatory sequence of the transcription in a cell, b) allowing said vector to integrate into the genome of said cell by non-homologous recombination, c) allowing the overexpression of an endogenous gene in said cell by upregulating said gene by said transcription regulatory sequence, d) selecting said cell for overexpression of said endogenous gene, and e) introducing said cell isolated and cloned in an animal under conditions that favor overexpression of said endogenous gene by said cell in vivo. 36.- A method for producing an expression product of an endogenous cellular gene in vivo, characterized in that it comprises: a) introducing a vector comprising a transcription regulatory sequence operably linked to an unpaired sequence of splice donor in a cell , b) allowing said vector to be integrated into the genome of said cell by non-homologous recombination, c) allowing the overexpression of an endogenous gene in said cell by upregulating said gene by said transcription regulatory sequence, d) selecting said cell for the overexpression of said endogenous gene, and e) introducing said cell isolated and cloned in an animal under conditions that favor the overexpression of said endogenous gene by said cell in vivo. 37.- A method for producing an expression product of an endogenous cellular gene, characterized in that it comprises: a) introducing a vector comprising a transcription regulatory sequence and one or more amplifiable markers in a cell, b) allowing said vector is integrated into the genome of said cell by non-homologous recombination, c) allowing overexpression of an endogenous gene in said cell by upregulating said gene by said transcription regulatory sequence, d) selecting said cell for overexpression of said gene endogenous, e) culturing said cell under conditions in which said vector and said endogenous gene are amplifiable in said cell, and (f) culturing said cell under conditions that favor the production of the expression product of said endogenous gene by said cell. 38.- The method according to claim 37, further characterized in that it comprises isolating said expression product. 39.- A method for overexpressing an endogenous gene in a cell in vivo, characterized in that it comprises: a) introducing a vector comprising a transcription regulatory sequence and one or more amplifiable markers in a cell, b) allowing said vector to be integrates into the genome of said cell by non-homologous recombination, c) allows overexpression of an endogenous gene in said cell by upregulating said gene by said transcription regulatory sequence, d) selecting said cell for overexpression of said endogenous gene , and e) introducing said isolated and cloned cell into an animal under conditions that favor overexpression of said endogenous gene by said cell in vivo. 40. The method according to any of claims 26, 28-33, 35-37 or 39, further characterized in that said transcription regulatory sequence is a promoter. 41. The method according to claim 40, further characterized in that said promoter is a viral promoter. 42. The method according to claim 41, further characterized in that said viral promoter is the cytomegalovirus immediate early promoter. 43. The method according to claim 40, further characterized in that said promoter is a non-viral promoter. 44. The method according to claim 40, further characterized in that said promoter is inducible. 45. The method according to any of claims 26, 28-33, 35-37 or 39, further characterized in that said transcription regulatory sequence is an enhancer. 46. The method according to claim 45, further characterized in that said enhancer is a viral enhancer. 47. The method according to claim 46, further characterized in that said viral enhancer is the immediate early enhancer of cytomegalovirus. 48. The method according to claim 45, further characterized in that said enhancer is a non-viral enhancer. 49. The method according to any of claims 26, 28-33, 35-37 or 39, further characterized in that it comprises introducing double-stranded breaks in the genomic DNA of said cell before, or simultaneously with, the integration of said vector. 50.- A cell produced by the method according to any of claims 26, 28-33, 35-37 or 39. 51.- The method according to any of claims 29-33, 35-37 or 39, further characterized in that said vector construction is linear. 52. A method for overexpressing an endogenous gene in a cell, characterized in that it comprises: a) introducing a vector comprising a regulatory sequence of the transcription in a cell, b) allowing said vector to be integrated into the genome of said cell by means of non-homologous recombination, c) allowing the overexpression of an endogenous gene in said cell by upregulating said gene by said transcription regulatory sequence, d) selecting said cell for overexpression of said endogenous gene, and e) culturing said cell in a medium free of serum. 53.- A method for producing an expression product of an endogenous cellular gene, characterized in that it comprises: a) introducing a vector comprising a transcription regulatory sequence in a cell, b) allowing said vector to be integrated into the genome of said cell by non-homologous recombination, c) allowing overexpression of an endogenous gene in said cell by upregulating said gene by said transcription regulatory sequence, d) selecting said cell for overexpression of said endogenous gene, e) cultivating said cell under conditions that favor the production of the expression product of said endogenous gene by said cell, and (f) isolating said expression product from a cell mass equivalent to 10 liters of cells at 10 4 cells / ml. 54.- A method for activating the expression of a gene, characterized in that it comprises: a) introducing a vector into the genome of a cell, said vector containing a regulatory sequence and an unpaired site of a splice donor and that lacks sequences of identification, and b) selecting said cell for the expression of a gene. 55. The method according to claim 54, further characterized in that it comprises isolating the cell that produces the activated protein. 56.- A method for activating the expression of a gene, characterized in that it comprises: a) integrating a vector in a cell through non-homologous recombination, said vector contains a regulatory sequence and an unpaired splice donor site; and b) select for non-homologous recombinant cells that express a gene, wherein said gene and the region towards the 5 'end of said gene lack homology with the vector. 57.- A method to increase the expression of a gene in a cell in situ, the phenotype of said gene is known, without making use of any sequence information of the gene, the method comprises the steps of: a) construct a vector that comprising a transcriptional regulatory sequence and an unpaired splice donor sequence, b) supplying vector copies to a plurality of cells, c) culturing the cells under conditions that allow non-homologous recombination events between the inserted vector and the genome of the cells, and d) selecting the recombinant cells by testing the phenotype to identify cells in which the expression of the gene has been increased. 58. The method according to claim 57, further characterized in that the phenotype is the production of a particular protein, and the test is carried out by testing the increased production of the protein. 59.- A method to increase the expression of a gene in a cell in situ, the phenotype of said gene is known, without making use of any sequence information of the gene, the method comprises the steps of: a) construct a vector that comprising a transcriptional regulatory sequence and an unpaired splice donor sequence, b) supplying vector copies to a plurality of cells, c) culturing the cells under conditions that increase the probability of non-homologous recombination events between the vector and the genome of the cells, and d) selecting the recombinant cells by testing the phenotype to identify cells in which the expression of the gene has been increased. 60.- A method for activating the expression of a gene in a cell in situ without making use of any sequence information of the gene, the method comprises the steps of: a) constructing a vector comprising a regulatory sequence of the transcription and a unpaired sequence of splice donor, b) integrate the vector by non-homologous recombination into at least 100,000 cells, and c) select the recombinant cells by testing the phenotype to identify cells in which the expression of the gene has been activated. 61.- An isolated cell comprising in its genome an inserted genetic construct, the genetic construct comprises a transcriptional regulatory sequence operably linked to an unpaired sequence of splice donor, wherein said construct is inserted into a gene or region towards the 5 'end of a gene and activates the expression of said gene, and wherein the gene and the region towards the 5' end of said gene, do not have nucleotide sequence homology with the genetic construct. 62. The cell according to claim 61, further characterized in that the integrated genetic construct additionally contains one or more amplifiable markers. 63.- An isolated cell comprising in its genome an inserted genetic construct, said genetic construct comprises a transcriptional regulatory sequence operably linked to an unpaired sequence of splice donor, wherein said construct does not have nucleotide sequence homology with said gene or regions towards the 5 'end of said gene. 64.- A method for activating the expression of a gene, characterized in that it comprises: a) constructing a vector comprising a regulatory sequence of the transcription and an unpaired sequence of a splice donor, b) introducing said vector into a cell, c ) culturing the cell under conditions that allow non-homologous recombination events between the inserted vector and the genome of the cells, and d) selecting the recombinant cells by testing the expression of a gene, wherein said gene and the region towards the end 'of said gene do not have nucleotide sequence homology with the vector. 65.- An isolated cell that includes an inserted genetic construct in its genome, the genetic construct comprises a transcriptional regulatory sequence operably linked to an unpaired splice donor sequence, wherein said genetic construct is inserted into a gene or region towards the 5 'end of a gene by non-homologous recombination, and wherein said genetic construct activates the expression of said gene. 66.- A method for increasing the expression of a gene, characterized in that it comprises: a) introducing a vector into the genome of a cell, said vector containing an enhancer sequence and one or more amplifiable markers and lacking identification sequences, and ) select said cell for the expression of a gene. 67.- The method according to claim 66, further characterized in that it comprises isolating the cell that produces the activated protein. 68. - A method for increasing the expression of a gene, characterized in that it comprises: a) integrating a vector in a cell by non-homologous recombination, said vector containing an enhancer sequence, and b) selecting for non-homologous recombinant cells that express a gene, in where said gene and the regions towards the 5 'end and towards the 3' end of said gene, in whose regions the enhancer is active, lack homology with the vector. 69.- A method to increase the expression of a gene of known phenotype in a cell in situ without making use of any sequence information of the gene, the method comprises the steps of: a) constructing a vector comprising an enhancer, b) provide copies of the vector to a plurality of cells, c) culture said cells under conditions that allow non-homologous recombination events between the vector and the cell genome, and d) select the recombinant cells by testing the phenotype to identify cells in the cells. which gene expression has been increased. The method according to claim 69, further characterized in that the phenotype is the production of a particular protein, and the test is carried out by testing the increased production of the protein. 71.- A method to increase the expression of a gene of known phenotype in a cell in situ without making use of any sequence information of the gene, the method comprises the steps of: a) building a vector comprising an enhancer, b) supplying vector copies to a plurality of cells, c) culturing said cells under conditions that increase the probability of non-homologous recombination events between the vector and the cell genome, and d) selecting the recombinant cells by testing the phenotype to identify cells in which the expression of the gene has been increased. 72.- A method to increase the expression of a gene in a cell in situ without making use of any sequence information of the gene, the method comprises the steps of: a) building a vector comprising an enhancer, b) integrating the vector by non-homologous recombination in at least 100,000 cells, and c) selecting the recombinant cells by testing the phenotype to identify cells in which the expression of the gene has been increased. 73.- A purified cell that includes in its genome, an inserted artificial genetic construct, the genetic construction comprises an effective enhancer in the cell line to increase the expression of a gene, the genetic construct inserted in a gene or the regions towards the end 5 'or towards the 3' end of a gene, wherein said enhancer is effective, the gene and the regions having no sequence homology in the genetic construct. The cell according to claim 73, further characterized in that the integrated genetic construct additionally contains one or more amplifiable markers. 75. - An isolated cell that includes in its genome an inserted artificial genetic construct, the genetic construct comprises an effective enhancer in the cell line to increase the expression of a gene, the genetic construct has no homology with any sequence in said gene or with the regions towards the 5 'end or towards the 3' end of said gene wherein said enhancer is effective. 76.- A method for increasing the expression of a gene, characterized in that it comprises: a) constructing a vector comprising an enhancer, b) introducing said vector into a cell, c) cultivating said cell under conditions that allow non-homologous recombination events between the inserted vector and the genome of the cell, and d) selecting the cell by testing the expression of a gene, wherein said gene and the regions towards the 5 'end and towards the 3' end of said gene, wherein said enhancer is effective, they have no homology to the vector. 77.- An isolated cell that includes an inserted genetic construct in its genome, the genetic construct comprises an effective enhancer in the cell line to activate the expression of an endogenous gene in said cell, the genetic construct inserted in a gene or the regions towards the 5 'end or towards the 3' end of a gene by recombination does not homologate. 78. The vector construction according to claim 7, further characterized in that said vector construction comprises one, two, three, four or five amplifiable markers. 79. - The vector construction according to claim 8, further characterized in that said vector construction comprises one, two, three, four or five amplifiable markers. 80.- The vector construction according to claim 78 or 79, further characterized in that said vector construction comprises an amplifiable marker. 81. The vector construction according to claim 78 or 79, further characterized in that said vector construction comprises two amplifiable markers. 82. The method according to any of claims 37, 39 or 66, further characterized in that said vector construction comprises one, two, three, four or five amplifiable markers. 83. The method according to claim 82, further characterized in that said vector construct comprises an amplifiable marker. 84. The method according to claim 82, further characterized in that said vector construction comprises two amplifiable markers. 85.- The cell according to claim 62 or 74, further characterized in that said integrated genetic construction comprises one, two, three, four or five amplifiable markers. 86.- The cell according to claim 85, further characterized in that said integrated genetic construct comprises an amplifiable marker. 87. The cell according to claim 85, further characterized in that said integrated genetic construct comprises two amplifiable markers. 88. The method according to any of claims 26, 28-33, 35-37, 39, 52 or 53, further characterized in that said endogenous gene codes for a transmembrane protein. 89. The method according to claim 23, further characterized in that said endogenous gene codes for a cellular transmembrane protein. 90. The method according to any of claims 54, 56, 57, 59, 60, 64, 66, 68, 69, 71, 72 or 76, further characterized in that said gene codes for a cellular transmembrane protein. 91. The method according to any of claims 35, 36 or 39, further characterized in that it comprises isolating and cloning said cell before introducing said cell into an animal. 92. The method according to any of claims 35, 36 or 39, further characterized in that said animal is a mammal. 93. The method according to claim 92, further characterized in that said mammal is a human. 94.- A method for identifying an endogenous gene that codes for a cellular integral membrane protein, characterized in that it comprises: a) introducing a vector comprising a regulatory sequence of the transcription in a cell, b) allowing said vector to integrate into the genome of said cell by non-homologous recombination, c) allowing overexpression of an endogenous gene in said cell by upregulating said gene by said transcription regulatory sequence, d) selecting said cell for overexpression of said endogenous gene, and e) characterizing said activated gene to determine its identity as a gene encoding a cellular integral membrane protein. 95. The method according to claim 94, further characterized in that said activated gene is isolated from said cell before said characterization. SUMMARY OF THE INVENTION The present invention generally relates to activating the expression of a gene or causing overexpression of a gene by in situ recombination methods; the invention also generally relates to methods for expressing an endogenous gene in a cell at levels higher than those normally found in the cell; In one embodiment of the invention, the expression of an endogenous gene is activated or increased after integration into the cell, by non-homologous or illegitimate recombination, of a regulatory sequence that activates the expression of a gene.; in another embodiment, the expression of the gene can also be increased by the joint integration of one or more amplifiable markers, and selected for increased copies of one or more amplifiable markers located in the integrated vector; the invention also provides methods for the identification and expression of genes that can not be discovered by current methods since the identification sequence for integration is not necessary; the invention further provides methods for the isolation of nucleic acid molecules (mainly cDNA molecules) that encode transmembrane proteins and for the isolation of cells expressing said transmembrane proteins which may be heterologous transmembrane proteins; In addition, the invention relates to isolated genes, gene products, nucleic acid molecules and compositions comprising said genes, gene products and nucleic acid molecules that can be used in a variety of therapeutic and diagnostic applications; therefore, by the present invention, endogenous genes, including those associated with diseases and human development, can be activated and isolated without prior knowledge of the sequence, structure, function or expression profile of the genes. P00 / 395F