WO2008085564A2 - Compositions and methods involving truncated recombinant seven g-protein coupled receptors - Google Patents

Abstract

Disclosed are methods for tracking the location of a cell and/or its progeny in a subject, such as a stem cell, that involve (a) obtaining a cell; (b) trans fecting the cell with an expresison construct comprising a first coding region encoding a first reporter comprising a truncated recombinant seven transmembrane G-protein associated receptor (GPCR) amino acid sequence operatively linked to a first promoter sequence; (c) introducing the cell to the subject; and (d) detecting the location of the cell in the subject by contacting the cell with a detectable moiety that binds to the truncated recombinant GPCR and imaging the detectable moiety using a non-invasive imaging technique. The cell, for example, may be a stem cell or an immune cell. Also disclosed are non-human transgenic animals whose genome comprises a nucleic acid encoding a truncated recombinant GPCR amino acid sequence. Also disclosed are methods of producing stem cells that express a truncated recombinant GPCR, comprising obtaining a transgenic animal of the present invention and isolating stem cells from the transgenic animal.

Description

DESCRIPTION

COMPOSITIONS AND METHODS INVOLVING TRUNCATED RECOMBINANT SEVEN G-PROTEIN COUPLED RECEPTORS

BACKGROUND OF THE INVENTION

This application claims the benefit of priority to U.S. Application Serial No.

60/845,934, filed September 20, 2006, the entire contents of which is hereby incorporated by reference in its entirety.

1. Field of the Invention

The present invention relates generally to the fields of cell biology, imaging, and molecular biology. More particularly, one aspect of the invention pertains to a method for tracking the location of a cell in a subject. In particular embodiments, the cell is a stem cell or an immune cell. The invention also generally pertains to methods for detecting the differentiation of a cell, such as a stem cell or an immune cell, in a subject.

2. Description of Related Art Stem cells are cells that have the ability to continually reproduce themselves while maintaining the capacity to give rise to other more specialized types of cells. They may be obtained from natural sources, or generated through artificial means such as nuclear transfer, cytoplasmic transfer, cell fusion, parthenogenesis and reprogramming. Isolated stem cells can give rise to many types of differentiated cells. Two main groups of stem cells include adult stem cells and embryonic stem cells.

Adult stem cells are undifferentiated, but are capable of differentiation into the cell types from the tissue that the adult stem cell originated. Exemplary sources of adult stem cells include the nervous system (McKay, 1997; Shihabuddin et al, 1999), bone marrow (Pittenger et al, 1999; Pittenger and Marshak, 2001), adipose tissue (Gronthos et al, 2001), dermis (Toma et al, 2001), pancreas, liver (Deutsch et al, 2001), umbilical cord (Rogers et al, 2004; Wang et al, 2004; Surbek et al, 2002), and placenta (Yen et al, 2005).

It is believed that stem cells of the adult type are also found in many other tissues, such as smooth muscle tissue, striated muscle tissue, cardiac muscle tissue, bone tissue, bone spongy tissue, cartilage tissue, pancreatic ductal tissue, spleen tissue, thymus tissue, tonsil tissue, Peyer's patch tissue, lymph nodes tissue, thyroid tissue, epidermis tissue, dermis tissue, subcutaneous tissue, heart tissue, lung tissue, vascular tissue, endothelial tissue, blood cells, bladder tissue, kidney tissue, digestive tract tissue, esophagus tissue, stomach tissue, small intestine tissue, large intestine tissue, adipose tissue, uterus tissue, eye tissue, lung tissue, testicular tissue, ovarian tissue, prostate tissue, connective tissue, endocrine tissue, and mesentery tissue.

Stem cells can be applied in the treatment of many diseases. For example, stem cells derived from bone marrow cells are an established therapy in patients with hematological malignancies. Stem cells have been utilized in the treatment of solid tumors. Stem cells have also been used in "reprogramming" the immune system to induce an antigen-specific state of non-responsiveness called tolerance. Specifically, stem cells have been used to induce donor- specific tolerance during allogeneic or xenogeneic transplantation, and to induce tolerance in situations of autoimmunity.

One of the main therapeutic uses for stem cells is in the area of regenerative medicine. The concept of regenerative medicine is to restore or enhance the ability of tissues to self- organize and heal themselves following endogenous or exogenous injury. For example, for over twenty years, it has been known that bone marrow-derived cells give rise to both hematopoietic cells and stroma cells. Bone marrow cells have been shown to generate muscle fibers (Ferrari et ah, 1998), cardiomyocytes (Orlic et ah, 2001), microglia and astroglia (Eglitis and Mezey, 1997), and other tissues. Repopulation of diseases organs is implied in studies where dystrophin-expressing cells were found after transplantation in a mouse model of muscular dystrophy (Gussoni et ah, 1999). Evidence of transdifferentiation has also been found in humans. For example, apparently donor-derived hepatocytes have been found in liver biopsies after bone marrow and peripheral blood transplantation (Korbling et α/., 2002).

Stem cells have also been applied in the treatment of neurological deficiencies in a variety of situations. For example, administration of fetal stem cells into the striatal area of

Parkinson's disease patients results in the generation of dopaminergic neurons that can reinnervate the striatum, restore regulated dopamine release and movement-related frontal cortical activation, and result in observable clinical benefit (Lindvall et al, 2004).

Other cells that have been introduced into a subject for therapy include immune cells. For example, immune cells have been applied in the treatment of cancer. An immune cell is defined herein to refer to a cell that recognizes and responds against microorganisms, viruses, and substances recognized as foreign and potentially harmful to the body. Examples of immune cells include T cells and B cells.

Methods of following stem cell trafficking following administration of stem cells into a subject, however, are exceedingly limited. In an experimental autoimmune encephalomyelitis model for multiple sclerosis, Costa et al (2001) transferred myelin basic protein-specific CD4+ T cells that were transduced to express IL- 12 p40 and luciferase. In vivo, luciferase was used to demonstrate trafficking to the central nervous system. However, luciferase, as well as other light-based imaging systems, such as GFP, do not significantly penetrate human tissue. They are therefore not currently feasible for assessing stem cell trafficking in humans.

In another system, using positron emission tomography (PET), Koehne et al (2003) demonstrated in vivo that Epstein-Barr virus (EBV)-specific T cells expressing herpes simplex virus- 1 thymidine kinase (HSV-TK) selectively traffic to EBV+ tumors expressing the T cells' restricting HLA allele. Furthermore, these T cells retain their capacity to eliminate targeted tumors. Capitalizing on sequential imaging, Dubey et al. (2003) demonstrated antigen specific localization of T cells expressing HSV-TK to tumors induced by murine sarcoma virus/Moloney murine leukemia virus (M-MS V/M-MuL V). HSV-TK, however, is not an optimal alternative for following stem cell or immune cell trafficking because it is known to induce an immune reaction (Berger et al, 2006; Perez-Cruet et al, 1994; Ramesh et al, 1996; Rainov et al, 2000).

The sodium/iodide symporter (NIS) has been used to follow transplantation of myoblasts (Vadysirisack et al, 2006). The degree of NIS expression is not the sole reason for dictating the degree of radioiodide uptake, and the degree of sodium iodide uptake is saturable and therefore not linear beyond a certain degree of expression; further the maximal radioiodide uptake induced by NIS gene transfer differs among different cells (Vadysirisack et al, 2006). The latter in particular and the fact that sodium iodide cell-surface trafficking is susceptible to disruption potentially could have a significant impact on detection when cells differentiate/fuse or in the case of lymphocytes, become activated (Vadysirisack et al, 2006). The findings also suggest that quantification of NIS expression may be limited to a narrow range of expression. Further, by its action as a pump, NIS may alter cellular homeostasis, thus, it is not an optimal alternative for following stem cell or immune cell trafficking. Thus, there is the need for improved methods for imaging stem cells and following stem cell trafficking that (1) can be effectively imaged in a subject; (2) do not adversely alter stem cell function and differentiation; and (3) are not associated with significant side effects.

Seven G protein-coupled receptors (GPCRs) are a large family of transmembrane receptors with seven transmembrane domains that participate in the transduction of an extracellular signal into an intracellular signal. GPCRs are also known as seven transmembrane receptors, 7TM receptors, and heptahelical receptors. GPCR family members are involved in all types of stimulus-response pathways. The diversity of functions is highlighted by the wide range of ligands recognized GPCRs, from photons (rhodopsin) to small molecules (histamine receptors) to proteins (chemokine receptors).

Somatostatin receptor (SSTR), belongs to the family of G protein-coupled receptors with seven transmembrane domains. SSTR2 can serve as a reporter of gene expression that can be quantified in vivo (Yang et al, 2005). Somatostatin receptors are over-expressed on a variety of tumors (John et al, 1996), and somatostatin receptor imaging can identify a variety of neuroendocrine malignancies, including carcinoid, islet cell tumor, pheochromocytoma, paraganglioma, small-cell lung cancer, and medullary thyroid cancer (Termanini et al, 1997; Lamberts et al., 2001; Kwekkeboom et al, 2000). For imaging, radiopharmaceutical analogs of the naturally occurring ligand, somatostatin, are used. Upon activation, somatostatin initiates a variety of signaling events that affect cellular functions such as secretion, chemotaxis, and growth suppression. These affects have been exploited using therapeutic analogs of somatostatin, for example, to ameliorate or prevent carcinoid syndrome (Nikou et al., 2005; Ducreux et al., 2000; Wymenga et al., 1999).

For somatostatin, there are six receptor types, 1, 2A and 2B, 3, 4, and 5. Types 2A and 2B are alternate splice variants that are identical, except that type 2A has a longer intracytoplasmic carboxy-terminus (Petersenn et al., 1999). Upon activation, SSTR2 regulates signaling such as cAMP (Schwartkop et al., 1999) and cGMP production. The latter appears to regulate cell proliferation (Lopez et al., 2001).

Among the receptor subtypes, human type 2 (SSTR2; Kluxen et al., 1992; Bell et al.

1993; Panetta et al., 1994; O 'Carroll et al., 1993; Yamada et al., 1992) has the highest affinity for the most common clinically used somatostatin imaging analog, l l llndium- labeled octreotide. This radiopharmaceutical and 99mTc-labeled analogs are used in clinical practice to detect tumors that endogenously over-express somatostatin receptors (John et al. , 1996) and have been used in animals models to image tumors that express exogenously introduced SSTR2, for example, as a reporter gene (Kundra et al., 2002).

Expression of SSTR2 can be localized and quantified in vivo using 111-Indium- labeled octreotide (Yang et al., 2005). For evaluating the effects of a ligand on signaling, the receptor itself may be assessed. Signaling by the somatostatin receptors may be mediated through the C-terminus of the receptor (Schwartkop et al., 1999; Hukovic et al., 1998).

Expression of GPCRs, such as SSTR2, for following the trafficking, differentiation, or localization of stem cells or immune cells has not been previously described. In particular

GPCR's such as SSTR2 that are limited in signaling and in affecting phenotypic change have not been described for following trafficking, viability, trans/differentiation or fusion, localization, or expression of linked gene products of/by cells and their progeny in vivo using non-invasive or invasive imaging.

Methods for following the trafficking and differentiation of stem cells are thus exceedingly limited. The time-course of engraftment and whether functional differentiated cells are produced after stem cell transplantation remains unclear. It is possible that stem cells may engraft transiently into a diseased organ, engraft and not differentiate, or engraft and differentiate. To fully exploit stem cells as a therapy, one needs methods to follow the location of stem cells or immune cells following introduction of the cells into a subject, and determine the degree of engraftment and transdifferentiation or fusion. SUMMARY OF THE INVENTION

The inventor has identified novel methods of following the trafficking and differentiation of cells, such as stem cells or immune cells, in a subject. These methods involve obtaining a cell and transferring into the cell a nucleic acid that encodes a full length or truncated recombinant GPCR, or a GPCR amino acid sequence. Further, the inventor has found that truncation of the recombinant GPCR results in a receptor that can bind a ligand, but which is signaling defective and/or has altered internalization. This feature allows for the imaging of cells and limits effects on cellular signaling, function, or differentiation.

The present invention generally pertains to a method for tracking the location of a cell in a subject, involving: (a) obtaining a cell; (b) transferring into the cell an expression construct that includes a first coding region encoding a first reporter that includes a truncated recombinant seven transmembrane G-protein associated receptor (GPCR) amino acid sequence operatively linked to a first promoter sequence; (c) contacting the cell with a detectable moiety that binds to the first reporter; (d) introducing the cell to the subject; and (e) imaging the detectable moiety using an imaging technique.

In some embodiments, the cells are contacted with a detectable moiety after the cells have been introduced into a subject. In some embodiments, the method for tracking the location of a cell in a subject can be further defined as a method for tracking the location of progeny of the cell in a subject. For example, following transfer into a stem cell of an expression construct that encodes a truncated recombinant GPCR amino acid sequence, the expression construct becomes incorporated into the genome of the stem cell, and therefore the progeny of the stem cell would include a nucleic acid sequence that encodes the same truncated recombinant GPCR amino acid sequence. Thus, progeny of the stem cell can be detected and imaged using any of the methods set forth herein. In these embodiments, the method may further involve contacting progeny of the cell with a detectable moiety that binds to the first reporter that is encoded in the progeny.

The cell that is obtained can be any cell known to those of ordinary skill in the art, but in particular embodiments the cell is a stem cell or an immune cell. A "stem cell" generally refers to any cell that has the ability to divide for indefinite periods of time and to give rise to specialized cells.

For example, the stem cell can be an embryonic stem cell, a somatic stem cell, a germ stem cell, an epidermal stem cell, or a tissue-specific stem cell. Examples of tissue-specific stem cells include a cancer stem cell, an adult neural stem cell, a human neuron, a human oligodendrocyte, a human astrocyte, a human keratinocyte stem cell, a human keratinocyte transient amplifying cell, a human melanocyte stem cell, a human melanocyte, a human foreskin fibroblast, a human duct cell, a human pancreatic islet, a human pancreatic β-cell, a human adult renal stem cell, a human embryonic renal epithelial stem cell, a human kidney epithelial cell, a human hepatic oval cell, a human hepatocytes, a human bile duct epithelial cell, a human embryonic endodermal stem cell, a human adult hepatocyte stem cell (controversial as to existence), a human mammary epithelial stem cell, bone marrow-derived stem cell, a human lung fibroblasts, a human bronchial epithelial cell, a human alveolar type II pneumocyte, a human skeletal muscle stem cell (satellite cell), a human cardiomyocyte, bone marrow mesenchymal stem cell, simple squamous epithelial cell, descending aortic endothelial cell, aortic arch endothelial cell, aortic smooth muscle cell, limbal stem cell, corneal epithelial cell, CD34+ hematopoietic stem cell, mesenchymal stem cell, osteoblast (precursor is mesenchymal stem cell), peripheral blood mononuclear progenitor cell, osteoclast, stromal cell, a human splenic precursor stem cell, a human splenocyte, a human CD4+ T-cell, a human CD8+ T-cell, a human NK cell, a human monocyte, a human macrophage, a human dendritic cell, a human B-cell, goblet cell, pseudostriated ciliated columnar cell pseudostriated epithelium stratified epithelial cell, ciliated columnar cell, goblet cell, basal cell, cricopharyngeus muscle cell, oesophageal stem cell, oesophageal transit amplifying cell, female primary follicle, and male spermatogonium.

An "immune cell" is any cell associated with generation of an immune response, such as a monocyte, a granulocyte, or a lymphocyte. The granulocyte may be a neutrophil, a basophil, or an eosinophil. The lymphocyte may be a T cell, a B cell, or a NK cell. The immune cell may be a stem cell whose progeny includes any of the aforementioned cells associated with generation of an immune response.

The cell can be obtained from any source, both natural and artificial. In some embodiments, the cell is an autologous cell. For example, the cell may be a stem cell obtained from a subject, wherein the cell is reintroduced into the subject following the transfer into the cell of the expression construct that comprises a coding region encoding a truncated recombinant GPCR. In other embodiments, the cell is an allogeneic cell, or a cell obtained from a subject that is distinct from the subject to whom the cell is introduced, but from the same species. In still further embodiments, the cell is a xenogeneic stem cell, or a cell from a different species than the recipient subject. In embodiments of the present invention, the first reporter includes a truncated recombinant GPCR amino acid sequence. The GPCR can be truncated at either the N- terminus or the C-terminus. In some embodiments, there is a truncation at both the N- terminus and the C-terminus. In particular embodiments, the recombinant GPCR has a C- terminal deletion. In certain embodiments, the truncation of the GPCR results in a GPCR that has altered signaling including is signaling defective, has altered internalization, or a combination thereof. Altered signaling includes an increase in signaling, a decrease in signaling, or an inciting of signaling pathways different from those incited by the wild type receptor.

The GPCR can be any GPCR known to those of ordinary skill in the art. For example, the GPCR may be an acetylcholine receptor: Ml, M2, M3, M4, or M5; adenosine receptor: Al; A2A; A2B; or A3; adrenoceptors: alphalA, alphalB, alphalD, alpha2A, alpha2B, alpha2C betal, beta2, or beta3; angiotensin receptors: ATI, or AT2; bombesin receptors: BBl, BB2, or BB3; bradykinin receptors: Bl, B2, calcitonin, Ainilin, CGRP, or adrenomedullin receptors; cannabinoid receptors: CBl, or CB2; chemokine receptors: CCRl, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCRlO, CXCRl, CXCR2, CXCR3, CXCR4, CXCR5, CX3CR1, or XCRl; chemotactic receptors : C3a, C5a, or fMLP; cholecystokinin and gastrin receptors: CCKl, or CCK2; corticotropin-releasing factor receptors: CRFl, or CRF2; dopamine receptors: Dl, D2, D3, D4, or D5; endothelin receptors: ET(A) or ET(B); galanin receptors: GALl, GAL2, or GAL3; glutamate receptors: mgll, mgl2, mgl3, mgl4, mgl5, mglβ, mgl7, or mgl8; glycoprotein hormone receptors: FSH, LSH, or TSH; histamine receptors: Hl, H2, H3, or H4; 5-HT receptors: 5-HT1A, 5-HT1B, 5- HTlD, 5-HT1B, 5-HT1F, 5HT2A, 5-HT2F, 5-HT2C, 5-HT3, 5-HT4, 5-HT5A, 5-HT5B, 5- HT6, or 5-HT7; leukotriene receptors: BLT, CysLTl, or CysLT2; lysophospholipid receptors: edgl, edg2, edg3, or edg4; melanocorlin receptors: MCl; MC2; MC3; MC4, or MC5; melatonin receptors: MTl, MT2, or MT3; neuropeptide Y receptors: Yl, Y2, Y4, Y5, or Y6; neurotension receptors: NTSl, or NTS2; opioids: DOP, KOP, MOP, or NOP; P2Y receptors: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, or P2Y12); peroxisome proliferators: PPAR- alpha, PPAR-beta, or PPAR-gamma; prostanoid receptors: DP, FP, IP, TP, EPl, EP2, EP3, or EP4; protease-activated receptors: PARl, PAR2, PAR3, or PAR4; Somatostatin receptors: SSTRl, SSTR2, SSTR2A, SSTR3, SSTR4, or SSTR5; tachykinin receptors: NKl, NK2, or NK3; thyrotropin-releasing hormone receptors: TRHl, or TRH2; urotensin-II receptor; vasoactivate intestinal peptide or pituitary adenylate cyclase activating peptide receptors: VPACl, VPAC2, or PACl; or vasopressin or oxytocin receptors: Via, VIb, V2, or OT.

In particular embodiments of the present invention, the GPCR is a somatostatin receptor. For example, the somatostatin receptor may be a somatostatin receptor type 1, 2A and 2B, 3, 4, or 5. In further particular embodiments, the somatostatin receptor is a somatostatin receptor type 2A (SSTR2A). The SSTR2A may have altered signaling including signaling defective, have altered internalization, or a combination thereof. Information regarding somatostatin fusion proteins can be found in U.S. Patent App. Pub. No. 20020173626, herein specifically incorporated by reference.

The first promoter sequence can be any promoter sequence known to those of ordinary skill in the art. Promoter sequences are discussed in detail elsewhere in this specification. For example, the promoter sequence may be a function-specific promoter sequence, a constitutive promoter sequence, or a tissue-selective promoter sequence. In particular embodiments, the first promoter sequence is a function-specific promoter sequence. A "function specific promoter sequence" is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled, wherein the sequence is active in cells and whose products perform a particular function of interest. Examples of tissue selective promoter sequences include an insulin promoter sequence, T cell receptor promoter sequence, immunoglobulin promoter sequence, hormone or paracrine promoters such as vascular endothelial growth factor promoter sequences, structural protein promoters such as a dystrophin promoter sequence, intracellular component such as fat or melanin promoter sequences, or extracellular component such as cartilage promoter sequences. Other examples include a pBROAD promoter sequence, a c-fos promoter sequence, a c-HA-ras promoter sequence, an intercellular adhesion molecule 2 promoter sequence, and a platelet-derived growth factor (PDGF) promoter sequence.

The first promoter sequence may also be a constitutive promoter sequence. A "constitutive promoter sequence" is defined herein to refer to a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled, wherein the sequence is active in cells of most any lineage. For example, the constitutive promoter sequence may be a beta-actin promoter sequence, an elastase I promoter sequence, a metallothionein (MTII) promoter sequence, a 5 S ribosomal promoter sequence, an Elastase promoter sequence, an Elastase I promoter sequence, a polyoma promoter sequence, a Cytomegalovirus promoter sequence, a retrovirus promoter sequence, a papilloma virus promoter sequence, a fibronectin promoter sequence, a ubiquitin promoter, an actin promoter, an elongation factor 1 alpha, an early growth factor response 1 , an eukaryotic initiation factor 4Al, a ferritin heavy chain, a ferritin light chain, a glyceraldehyde 3 -phosphate dehydrogenase, a glucose-regulated protein 78, a glucose-regulated protein 94, a heat shock protein 70, a heat shock protein 90, a beta-kinesin, a phosphoglycerate kinase, an ubiquitin B, a beta-actin, RNA virus promoter, DNA virus promoter, adenoviral promoter sequence, a baculoviral promoter sequence, a CMV promoter sequence, a parvovirus promoter sequence, a herpesvirus promoter sequence, a poxvirus promoter sequence, an adeno-associated virus promoter sequence, a semiliki forest virus promoter sequence, an SV40 promoter sequence, a vaccinia virus promoter sequence, a lentivirus promoter, a retrovirus promoter sequence, or a minimal viral promoter sequence. One of ordinary skill in the art would be familiar with these and other constitutive promoter sequences. In some embodiments, the constitutive promoter is a minimal viral promoter sequence. For example, the minimal viral promoter sequence may be a RNA virus promoter, DNA virus promoter, adenoviral promoter sequence, a baculoviral promoter sequence, a CMV promoter sequence, a parvovirus promoter sequence, a herpesvirus promoter sequence, a poxvirus promoter sequence, an adeno-associated virus promoter sequence, a semiliki forest virus promoter sequence, an SV40 promoter sequence, a vaccinia virus promoter sequence, a lentivirus promoter, or a retrovirus promoter sequence.

In further embodiments, the first promoter sequence is a tissue selective promoter sequence. A "tissue selective promoter sequence" is defined herein to refer to a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled, wherein the sequence is active in cells of a particular lineage or tissue type. For example, the tissue-selective promoter sequence may be a promoter sequence that is active in normal and/or diseased heart, lung, esophagus, muscle, intestine, breast, prostate, stomach, bladder, liver, spleen, pancreas, kidney, neurons, myocytes, leukocytes, immortalized cells, neoplastic cells, tumor cells, cancer cells, duodenum, jejunum, ileum, cecum, colon, rectum, salivary glands, gall bladder, urinary bladder, trachea, larynx, pharynx, aorta, arteries, capillaries, veins, thymus, lymph nodes, , bone marrow, pituitary gland, thyroid gland, parathyroid glands, adrenal glands, brain, cerebrum, cerebellum, medulla, pons, spinal cord, nerves, skeletal muscle, smooth muscle, bone, testes, epidiymides, prostate, seminal vesicles, penis, ovaries, uterus, mammary glands, vagina, skin, eyes, or optic nerve.

In some embodiments, the tissue-selective promoter sequence is an hTR promoter sequence, a hTERT promoter sequence, a CEA promoter sequence, a PSA promoter sequence promoter sequence, a probasin promoter sequence, a ARR2PB promoter sequence, an AFP promoter sequence, a MUC-I promoter sequence, a MUC-4 promoter sequence, a mucin-like glycoprotein promoter sequence, a C-erbB2/neu oncogene promoter sequence, a cyclo- oxygenase promoter sequence, a E2F transcription factor 1 promoter sequence, a tyrosinase related protein promoter sequence, a tyrosinase promoter sequence, a survivin promoter sequence, a Tcfl -alpha promoter sequence, a Ras promoter sequence, a Raf promoter sequence, a cyclin E promoter sequence, a Cdc25A promoter sequence, a HK II promoter sequence, a KRT 19 promoter sequence, a TFFl promoter sequence, a SELlL promoter sequence, or a CEL promoter sequence. Other examples of tissue-selective promoter sequences include an immunoglobulin heavy chain promoter sequence, an immunoglobulin light chain promoter sequence, a T-cell receptor promoter sequence, an HLA DQ a promoter sequence, an HLA DQ beta promoter sequence, a beta-interferon promoter sequence, an interleukin-2 promoter sequence, an interleukin-2 receptor promoter sequence, an MHC Class II 5 promoter sequence, an MHC Class II HLA-Dra promoter sequence, , a muscle creatine kinase (MCK) promoter sequence, a prealbumin (transthyretin) promoter sequence, an albumin promoter sequence, an alpha- fetoprotein promoter sequence, a gamma-globin promoter sequence, a beta-globin promoter sequence, a, an insulin promoter sequence, a neural cell adhesion molecule (NCAM) promoter sequence, an alpha- 1 -antitrypsin promoter sequence, a growth hormone promoter sequence, a human serum amyoid A (SAA) promoter sequence, a troponin I (TN I) promoter sequence, a Duchenne Muscular Dystrophy promoter sequence, an SV40 promoter sequence, a Hepatitis B virus promoter sequence, a Gibbon Ape Leukemia Virus promoter sequence, a somatostatin receptor promoter sequence, a human CD4 promoter sequence, a human alpha- lactalbumin promoter sequence, a human Y promoter sequence, an alpha fetoprotein promoter sequence, a monocyte receptor for bacterial LPS promoter sequence, a leukocyte common antigen promoter sequence, a Desmin promoter sequence, a VEGF receptor promoter sequence, a glial fibrillary acidic protein promoter sequence, an interferon beta promoter sequence, a myoglobin promoter sequence, an osteocalcin 2 promoter sequence, a prostate specific antigen promoter sequence, a prostate specific membrane antigen promoter sequence, a surfactant protein B promoter sequence, a Synapsin promoter sequence, a tyrosinase related protein promoter sequence, a tyrosinase promoter sequence, a functional hybrid, functional portion, or a combination of any of the aforementioned promoter sequences. Some promoters may be both lineage specific and functional (e.g., albumin). Other examples of promoter sequences include a collagenase promoter sequence, an

H2B (TH2B) histone promoter sequence, a type I collagen promoter sequence, a GRP94 promoter sequence, a GRP78 promoter sequence, an other glucose-regulated protein promoter sequence, a Human Immunodeficiency Virus promoter sequence, a human LIMK2 gene promoter sequence, a murine epididymal retinoic acid-binding gene promoter sequence, a mouse alpha2 (XI) collagen promoter sequence, a DlA dopamine receptor promoter sequence, an insulin-like growth factor II promoter sequence, a human platelet endothelial cell adhesion molecule- 1 promoter sequence, a 7SL promoter sequence, a human MRP-7-2 promoter sequence, a leukosialin promoter sequence, a Sialophorin promoter sequence, a Macrosialin or human analogue of macrosialin promoter sequence, and an Endoglin promoter sequence.

In some embodiments, the nucleic acid includes more than one coding region. For example, the nucleic acid may include a second coding region. Preferably, a second promoter sequence is operatively linked to the second coding region. In some embodiments, the first coding region and the second coding region are linked by an IRES or a bidirectional promoter sequence. A "bidirectional promoter sequence" refers to control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription of both the first and the second coding region are controlled. One of ordinary skill in the art would be familiar with bidirectional promoter sequences, such as those set forth in Trinklein et ah,

2004 and http:// www.stanford.eduMdschroed/bidirectional/index.shtml.)

The first promoter sequence and the second promoter sequence may be promoter sequences that are individually selected from the group consisting of a constitutive promoter sequence, a tissue-specific promoter sequence, a lineage-specific promoter, and a function- specific promoter sequence. For example, the first promoter sequence and the second promoter sequence may be of the same type {e.g., both constitutive promoter sequences) or may differ in type {e.g., first promoter sequence is a constitutive promoter sequence, and second promoter sequence is a tissue-specific promoter sequence).

The second coding region may encode, for example, a reporter sequence, a therapeutic gene, or signaling sequence, or a trafficking sequence. The term "reporter,"

"reporter gene" or "reporter sequence" as used herein refers to any genetic sequence or encoded polypeptide sequence that is detectable and distinguishable from other genetic sequences or encoded polypeptides present in cells. A "therapeutic gene" as used herein refers to any genetic sequence or encoding polynucleotide sequence that is known or suspected to be of benefit in the treatment or prevention of disease in a subject. A "signaling sequence" is defined herein to refer to any genetic sequence or encoded polynucleotide sequence that is involved in signal transduction or cell differentiation. A "trafficking sequence" as used herein refers to any genetic sequence or encoded polypeptide sequence that is involved in the transit of cells from one site in a subject to a different site in the subject. Reporter sequences, therapeutic genes, signaling sequences, and trafficking sequences are well-known to those of ordinary skill in the art, and are discussed at length elsewhere in this specification. In further embodiments, the nucleic acid further includes a third coding region. Preferably, the third coding region is operatively linked to a third promoter sequence. The first coding region, the second coding region, and the third coding region may either be independent or operably linked by one or more IRES or bidirectional promoter sequences. The first promoter sequence, the second promoter sequence, and the third promoter sequence may be individually selected from the group consisting of a constitutive promoter sequence, a tissue-specific promoter sequence, a lineage-specific promoter, and a function-specific promoter sequence. As discussed above, the promoter sequences may be of the same type or be of distinct types. In some embodiments, the first coding region, the second coding region, and the third coding region are individually selected from the group consisting of a reporter sequence, a therapeutic gene, or signaling sequence, or a trafficking sequence. In a specific embodiment, the first promoter sequence is a constitutive promoter sequence, the second promoter sequence is a tissue-specific promoter sequence, and the third promoter sequence is a lineage specific promoter sequence or a function-specific promoter sequence.

In some embodiments, the method further comprises sorting of the cell from other cells following the transfer of the expression construct. "Sorting" refers to separation of a cell containing the expression construct from other cells that do not contain the expression construct. Sorting can be performed by any method known to those of ordinary skill in the art, and may rely on the presence of the encoded truncated recombinant GPCR. For example, sorting may be performed by fluorescence activated cell sorting (FACS), column chromatography, and/or magnetic resonance beads.

Introducing the cell to the subject can be by any method known to those of ordinary skill in the art. For example, introducing the cell to the subject may involve intravenous administration, intracardiac administration, intradermal administration, intralesional administration, intrathecal administration, intracranial administration, intrapericardial administration, intraumbilical administration, intraocular administration, intraarterial administration, intraperitoneal administration, intraosseous administration, intrahemmorhage administration, intratrauma administration, intratumor administration, subcutaneous administration, intramuscular administration, intravitreous administration, direct injection into a normal organ, direct injection into a diseased organ, topical administration, or any other method of local or systemic administration known to those of ordinary skill in the art. In some embodiments, the method for tracking the location of a cell in a subject further comprises detecting expression of the first reporter by assaying for an association between the reporter expressed by the cell and a detectable moiety. For example, the association between the cell and the detectable moiety comprises binding of the detectable moiety by the cell, binding of a ligand operably coupled to the detectable moiety by the cell, cellular uptake of the detectable moiety, or cellular uptake of a ligand operably coupled to the detectable moiety.

A "detectable moiety" is defined herein to refer to any molecule or agent that can emit a signal that is detectable by imaging. For example, the detectable moiety may be a protein, a radioisotope, a fluorophore, a visible light emitting fluorophore, a near infrared light emitting fluorophore, infrared light emitting fluorophore, a metal, a ferromagnetic substance, a paramagnetic substance, a superparamagnetic substance, an electromagnetic emitting substance, a substance with a specific MR spectroscopic signature, an X-ray absorbing or reflecting substance, or a sound altering substance. In certain particular embodiments, the detectable moiety is a radioisotope. In particular embodiments, the detectable moiety is 111- In octreotide.

In further embodiments, the detectable moiety is operably coupled to a ligand that specifically binds the reporter. A "ligand" is defined herein to refer to an ion, a peptide, a oligonucleotide, a molecule, or a molecular group that binds to another chemical entity or polypeptide to form a larger complex. For example, in some embodiments, the ligand is a nucleic acid, such as a DNA molecule or an RNA molecule, a protein, a polypeptide, a peptide, an antibody, an antibody fragment, or a small molecule. In particular embodiments, the detectable moiety is a small molecule. In some embodiments, the detectable moiety is a near infrared light-emitting fluorophore. In some embodiments, the nucleic acid further encodes a protein tag fused to the N- terminal end or C-terminal end of the truncated GPCR amino acid sequence. The protein tag may or may not have enzymatic activity. In embodiments wherein the protein tag has enzymatic activity, the protein tag may be, for example, hemagglutinin A, beta-galactosidase, thymidine kinase, transferrin, myc-tag, VP 16, (His)₆-tag, FLAG, or chloramphenicol acetyl transferase. Contacting the cell with a detectable moiety may occur either prior to or after the cell is introduced into the subject. In particular embodiments, the cell is contacted with a detectable moiety prior to introduction of the cell into the subject.

The expression construct that is transferred into the cell may or may not be comprised in a delivery vehicle. A "delivery vehicle" is defined herein to refer an entity that associates with a nucleic acid and mediates the transfer of the nucleic acid into a cell. Any delivery vehicle is contemplated by the present invention. For example, the delivery vehicle may include but is not limited to a polypeptide, a lipid, a liposome, lipofectamine, a plasmid, a viral vector, a phage, a polyamino acid such as polylysine, a prokaryotic cell, or a eukaryotic cell.

In some embodiments, for example, the expression construct is comprised in a delivery vehicle, and transferring the expression construct into the cell comprises contacting the cell with the delivery vehicle. In particular embodiments, the delivery vehicle is a viral vector. The viral vector can be any viral vector known to those of ordinary skill in the art. For example, the viral vector may be a lentiviral vector, a baculovirus vector, a parvovirus vector, a semiliki forest virus vector, a Sindbis virus vector, a lentivirus vector, a retroviral vector, a vaccinia viral vector, an adeno-associated viral vector, a picornavirus vecctor, an alphavirus vector, or a poxviral vector. In some embodiments, the viral vector is a lentiviral vector. Transferring the expression construct into the cell can be by any method known to those of ordinary skill in the art. For example, transferring the expression construct may involve performing electroporation or nucleofection of the cell in the presence of the expression construct.

In some embodiments of the present invention, the cells are contacting with a detectable moiety that binds to the first reporter prior to introducing the cells into the subject, and in other embodiments, the cells are contacted with the detectable moiety that binds to the first reporter after the cells are introduced into the subject.

Any imaging technique known to those of ordinary skill in the art can be applied in imaging the detectable moiety. In some embodiments, for example, the imaging technique is an invasive imaging technique. An "invasive imaging technique" is defined herein to refer to any imaging technique that involves removal of tissue from a subject or insertion of a medical device into a subject. Invasive imaging techniques may involve, for example, performance of a biopsy of tissue in conjunction with an imaging technique such as fluorescence microscopy, or insertion of a catheter or endoscope into a subject for purposes of imaging.

In particular embodiments, the imaging technique is a non-invasive imaging technique. A "non-invasive imaging technique" is defined herein as an imaging technique that does not involve removal of tissue from a subject or insertion of a medical device into a subject. One of ordinary skill in the art would be familiar with non-invasive imaging techniques. Examples include MRI, MR spectroscopy, radiography, CT, ultrasound, planar gamma camera imaging, SPECT, PET, other nuclear medicine-based imaging, optical imaging using visible light, optical imaging using luciferase, optical imaging using a fluorophore, other optical imaging, imaging using near infrared light, and imaging using infrared light.

In some embodiments, the method for tracking the location of a cell in a subject may be further defined as a method for treating a subject with disease. Thus, for example, the cell to be tracked is a stem cell, and the stem cell is introduced into the subject for the purpose of treating a disease. The disease can be any disease known to those of ordinary skill in the art. For example, the disease may be a hyperproliferative disease, an infectious disease, an inflammatory disease, a degenerative disease, a congenital disease, a genetic disease, an immunological disease, trauma, poisoning, or a disease associated with toxicity. In particular embodiments, the disease is a hyperproliferative disease. The hyperproliferative disease may be benign or malignant. In particular embodiments, the hyperproliferative disease is cancer. The cancer may be any type of cancer. For example, the cancer may be breast cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, colon cancer, renal cancer, skin cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, uterine cancer, lymphatic cancer, stomach cancer, pancreatic cancer, testicular cancer, lymphoma, or leukemia. In further embodiments, the disease is type I diabetes or type II diabetes.

In still further embodiments, the disease is cardiovascular disease. For example, the cardiovascular disease may be cardiomyopathy, ischemic cardiac disease, congestive heart failure, congenital cardiac disease, traumatic cardiac disease, toxic cardiac disease, pericarditis, or genetic cardiac disease. Alternatively, the disease may be a neurological disease, such as Parkinson's disease, Alzeimer disease, amyotrophic lateral sclerosis, or multiple sclerosis. The neurological disease may be a neurodegenerative disease, spinal cord disease, traumatic neurological disease, infectious disease, or inflammatory disease. The disease may also be an immunological disease, such as transplant rejection, autoimmune disease, immune complex disease, vasculitis, or HIV infection.

In certain embodiments, the method for tracking the location of a cell and/or its progeny in a subject is further defined as a method of assessing the viability of a cell and/or its progeny in a subject. For example, the expression construct may include a first coding region encoding a reporter comprising a truncated recombinant GPCR amino acid sequence operatively linked to a constitutive promoter sequence. Imaging a detectable moiety that binds to the truncated recombinant GPCR sequence would provide an indicator not only on location, but of cell viability.

In further embodiments, the method for tracking the location of a cell in a subject may further be defined as a method for assessing the trans/differentiation or fusion of a stem cell and/or its progeny in a subject. For example, the expression construct may include a first coding region encoding a reporter comprising a truncated recombinant GPCR amino acid sequence operatively linked to a constitutive promoter sequence. Imaging a detectable moiety that binds to the truncated recombinant GPCR sequence would provide an indicator not only on location, but of cell viability. Imaging of a second coding region encoding a reporter operatively coupled to a second promoter sequence, such as a tissue-selective promoter sequence, a lineage-specific promoter sequence, or a function-specific promoter sequence, would provide evidence of differentiation of a stem cell and/ or its progeny in the subject, as the second coding region would only be encoded under conditions associated with some extent of cell differentiation.

In some embodiments, the method for tracking the location of a cell in a subject is further defined as a method for tracking the location of a tissue that is transplanted into a subject. The tissue can be any tissue suitable for transplantation. For example, the tissue may be heart tissue, islet cell tissue, or tissue from any organ that expresses a recombinant truncated GPCR. In these embodiments, introducing the cell into the subject is further defined as transplanting the tissue into the subject. The transplanted tissue may comprise one or more cells that include a reporter encoding a truncated recombinant GPCR amino acid sequence. The tissue may be made to differentiation into tissues of organs (e.g., embryonic stem cells into hear tissue) in vitro or in vivo, and then be transplanted into another animal. The truncated GPCR reporter may be used, for example, to track viability, incorporation, and trafficking of cells that may migrate from the graft. This is important for tissue engineering, such as generation of a new external ear or liver. One embodiment of the present invention is a method for tracking the location of a stem cell and/or its progeny in a human subject, which involves (a) obtaining a stem cell; (b) transfecting the stem cell with an expression construct comprising a first coding region encoding a first reporter comprising a recombinant somatostatin receptor truncated carboxy- terminal to amino acid 314 and operatively linked to a first promoter sequence; (c) introducing the stem cell to the subject; and (d) detecting the location of the stem cell and/or its progeny in the subject using an imaging technique to detect a detectable moiety that is bound to the truncated recombinant somatostatin receptor.

A further embodiment of the present invention is a method for detecting the differentiation of a stem cell and/or its progeny in a human subject, which involves: (a) obtaining a stem cell; (b) transfecting the stem cell with an expression construct comprising a first coding region encoding a first reporter comprising a somatostatin receptor truncated carboxy-terminal to amino acid 314 and operatively linked to a first promoter sequence; (c) introducing the stem cell to the subject; and (d) detecting the differentiation of the stem cell in the subject using an imaging technique to detect a detectable moiety that is bound to the truncated recombinant somatostatin receptor. The stem cell can be any of those stem cells discussed above and elsewhere in this specification. In particular embodiments, the stem cell is an immune progenitor cell. An immune progenitor cell is any cell whose progeny is an immune cell, such as any of the immune cells discussed above.

A further embodiment pertains to a method of tracking the location, lineage differentiation and function of a stem cell. For example, the stem cells can be (a) tracked to the pancreas using a constitutive promoter, (b) evaluated for trans/differention into islet cell lineage using an islet cell promoter such as Nkx2.2, and (c) assessed for function using a functional promoter such as the insulin promoter.

In particular embodiments, one or multiple types of immune cells are used, such as T- lymphocytes that bind a particular antigen or peripheral blood white blood cells, respectively.

The present invention also generally pertains to non-human transgenic animals whose genomes comprise a nucleic acid encoding a truncated recombinant GPCR amino acid sequence. "Transgenic animals" are non-human animals, preferably mammals, in which one or more of the cells include a transgene. Exemplary transgenic animals include primates, sheep, dogs, cats, rabbits, cows, goats, birds such as chickens, reptiles, amphibians, rodents such as rats and mice, etc. In certain embodiments, the transgenic animal is a mouse. The present invention also generally pertains to non-human transgenic animals whose genome comprises a nucleic acid encoding a recombinant GPCR receptor under the control of a heterologous promoter. A "heterologous promoter" is a promoter that is out of its naturally occurring context. The GPCR can be any GPCR known to those of ordinary skill in the art, including those set forth above. In particular embodiments, the GPCR is a somatostatin receptor, such as SSTR2. The heterologous promoter may be a constitutive promoter, a tissue-selective promoter, a lineage-specific promoter sequence, or a functional promoter.

In certain embodiments, the transgenic animal is a primate such as a monkey. A "transgene" is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops, and that remains in the genome of the mature animal. The nucleic acid encoding the truncated recombinant GPCR preferably is operably linked to a promoter. The promoter may be any promoter sequence, including any of those promoter sequences discussed above and elsewhere in this specification. In particular embodiments, a cell of the transgenic animal expresses a truncated recombinant seven transmembrane G- protein associated receptor (GPCR) amino acid sequence.

In particular embodiments, the truncated recombinant GPCR is a truncated somatostatin receptor. In further embodiments, the somatostatin receptor is a somatostatin receptor type 2A (SSTR2A) or a somatostatin receptor that is truncated carboxy terminal to amino acid 314. In some embodimets, the somatostatin receptor is a SSTR2delta314, which is a SSTR2A truncated carboxy terminal to amino acid 314.

The transgenic animal may be of any species, such as primates, sheep, dogs, cats, cows, goats, birds such as chickens, reptiles, amphibians, rodents such as rats and mice, etc. In certain embodiments, the transgenic animal is a mouse. In certain embodiments, the transgenic animal is a primate such as a monkey. Techniques pertaining to transgenic animals are well-known to those of ordinary skill in the art, and are reviewed in the specification below. Aspects of the present invention also generally pertain to a method of producing a cell that expresses a truncated recombinant GPCR amino acid sequence, that involves obtaining any of the transgenic animals set forth above and isolating one or more cells from the transgenic animal. In particular embodiments, the cell is a stem cell, an immune cell, or a cancer cell. The stem cell can be any stem cell, including any of the stem cells discussed above. In certain particular embodiments, the stem cell is an embryonic stem cell or a somatic stem cell.

In some particular embodiments, the truncated recombinant GPCR is a truncated somatostatin receptor. In particular embodiments, the somatostatin receptor is truncated carboxy terminal to amino acid 314. In some embodiments, the recombinant somatostatin receptor is a somatostatin receptor type 2A (SSTR2A).

The present invention also pertains to methods of producing a cell that expresses a recombinant somatostatin receptor amino acid sequence, comprising (a) obtaining a non- human transgenic animal whose genome includes a nucleic acid encoding a first reporter that includes a somatostatin receptor amino acid sequence and (b) isolating one or more cells from said transgenic animal. In some embodiments, the somatostatin receptor amino acid sequence is a somatostatin receptor type 2A (SSTR2A) amino acid sequence.

Other embodiments of the present invention pertain to methods for tracking the location of a cell in a subject, that involve (a) contacting a cell produced by any of the methods set forth herein with a detectable moiety that binds to the first reporter; (b) introducing the cell to the subject; and (c) imaging the detectable moiety using an imaging technique.

The nucleic acid encoding the truncated recombinant GPCR amino acid sequence or SSTR amino acid sequence preferably is operably linked to a promoter. The promoter may be any promoter sequence, including any of those promoter sequences discussed above and elsewhere in this specification. For example, the promoter may be a constitutive promoter, a tissue-selective promoter, a lineage-specific promoter sequence, or a functional promoter sequence. The nucleic acid encoding the truncated recombinant GPCR amino acid sequence may or may not further comprise a nucleic acid sequence encoding a protein tag. The protein tag may be any protein tag known to those of ordinary skill in the art. Examples are discussed elsewhere in this specification. In some embodiments, isolating the one or more cells from the transgenic animal involves sorting the cells. Sorting is performed to separate a cell expressing the encoded truncated recombinant GPCR from a cell that does not express the truncated recombinant GPCR. Sorting can be performed by any method known to those of ordinary skill in the art, including any of those methods discussed above and elsewhere in this specification. For example, sorting may involve FACS, separation using magnetic resonance beads, and/or column chromatography.

In some embodiments, tissues such as islets and organs that express the truncated GPCR reporter that are obtained from a transgenic animal may be transplanted into a subject, and the reporter may be used, for example, to track the viability, incorporation, and trafficking of cells that may migrate from the graft. In this regard, stem cells expressing the reporter may be made to differentiate into tissues or organs (e.g., embryonic stem cells into heart tissue) in vitro or in vivo, and then be transplanted into another animal. The reporter may be used, for example, to track viability, incorporation, and trafficking of cells that may migrate from the graft. This is important for tissue engineering, such as generation of a new external ear or liver. The reporter may be a GPCR such as SSTR, including SSTR2, or a GPCR with altered signaling and/or internalization such as a truncated SSTR reporter, including a truncated SSTR2 reporter. In some embodiments, the method is further defined as a method of treating a subject. The subject may have any disease, such as any of the diseases discussed above.

It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention.

Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention.

The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternative are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device and/or method being employed to determine the value. As used herein the specification, "a" or "an" may mean one or more, unless clearly indicated otherwise. As used herein in the claim(s), when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more. The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIGS. IA-I. Cell lines transfected with HA-wt SSTR2 or HA-SSTR2Δ314 express appropriate transcripts and similar levels of fusion protein. FIG. IA: Untransfected HT 1080 or HEK 293 cells do not express somatostatin receptors. No bands are seen in lanes SSTRl -5 of HT 1080 or HEK293 representing RT-PCR of cellular RNA. The quality of the same RNA for RT-PCR is confirmed by bands in the β-actin lanes (blank, no RNA). Lanes SSTRcDNA

1-5 demonstrate PCR of the expected sized bands for SSTR subtypes 1-5 using SSTR subtypes 1, 2, 3, 4, or 5 cDNA as templates. FIGS. IB-C: Transcripts of the appropriate size are detected by reverse-transcriptase polymerase chain reaction in HT1080 cells, as in FIG.

IB, or HEK 293 cells, as in FIG. 1C, transfected with HA-wt or HA-SSTR2Δ314. Lanes 1 and 3 primers for wt transcript, Lanes 2 and 4 primers for transcripts coding up to amino acid 314. FIGS. ID-G: Immunofluorescence targeting the HA domain demonstrate cell membrane localization of HA-wt or HA-SSTR2Δ314 fusion proteins; HT1080 clone expressing HA-wt, as shown in FIG. ID, or HA-SSTR2Δ314, as shown in FIG. IE, HEK293 clone expressing HA-wt, as shown in FIG. IF, or HA-SSTR2Δ314, as shown in FIG. IG. FIGS. IH-I: Equal protein expression is detected by ELISA targeting the HA domain in HT 1080, as shown in FIG. IH, or HEK 293, as shown in FIG. II, cell line pairs transfected with HA-wt or HA-SSTR2Δ314; whereas, no expression is seen in cells transfected with vector. Error bars represent SD of triplicate samples (*P < 0.05, Vector vs HA-wt SSTR2 or HA-SSTR2Δ314).

FIGS. 2A-E. HA-wild-type SSTR2 and HA-SSTR2Δ314 demonstrated similar binding to "^-octreotide. FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D: Representative Scatchard (left) and saturation (right) plots of ¹¹¹In-octreotide binding to membranes from HT 1080 cells transfected with HA-wt SSTR2, as in FIG. 2A, or HA-SSTR2Δ314, as in FIG. 2B, and of HEK293 cells transfected with HA-wt SSTR2, as in FIG. 2C, or HA-SSTR2Δ314, as in FIG. 2D. Binding affinity (Kd) and maximal binding values (B_max) values are average and SD of triplicate samples. FIG. 2E: The left graph in FIG. 2E shows the uptake of ^ulIn-octreotide by cells in vitro in the presence or absence of unlabeled somatostatin by HT1080 clones. The right graph in FIG. 2E shows the uptake of ^{l u}In-octreotide by cells in vitro in the presence or absence of unlabeled somatostatin by HEK 293 clones. The abbreviation "ss" refers to somatostatin; "V" refers to vector.

FIGS. 3A-D. HA-SSTR2Δ314 is signaling deficient for the cAMP pathway. FIG. 3A and FIG. 3C: Upon ligand binding, HA-wt SSTR2 decreases forskolin induced cAMP production in HT 1080 cells, as shown in FIG. 3 A, or in HEK 293 cells, as shown in FIG. 3C. *P < 0.05. FIG. 3B and FIG. 3D: Upon ligand binding, HA-SSTR2Δ314 does not decreases forskolin induced cAMP production when expressed in HT 1080 cells, as shown in FIG. 3B, or in HEK 293 cells, as shown in FIG. 3D.

FIGS. 4A-D. HA-SSTR2Δ314 is signaling deficient for the cGMP pathway. FIG. 4A and FIG. 4C: Upon ligand binding, HA-wt SSTR2 incites cGMP production when expressed in HT1080 cells, as shown in FIG. 4A, or in HEK 293 cells, as shown in FIG. 4C. (*P < 0.05, Phosphate-buffered saline (PBS) vs SS). FIG. 4B and FIG. 4D: Upon ligand binding, HA- SSTR2Δ314 does not incite cGMP production when expressed in HT1080 cells, as shown in FIG. 4B, or in HEK 293 cells, as shown in FIG. 4D. FIGS. 5A-D. HA-SSTR2Δ314 is deficient in inhibiting cell growth. Values on the abscissa are expressed as percent of color product of cells treated with Sandostatin compared to the same cells not exposed to Sandostatin. FIG. 5 A and FIG. 5C: Upon ligand binding, HA- wt SSTR2 decreases proliferation induced by serum in HT1080 cells, as shown in FIG. 5A, or in HEK 293 cells, as shown in FIG. 5C. *P < 0.05. FIG. 5B and FIG. 5D: Upon ligand binding, HA-SSTR2Δ314 does not decrease proliferation induced by serum in HT1080 cells, as shown in FIG. 5B, or in HEK 293 cells, as shown in FIG. 5D.

FIGS. 6A-B. HA-wt SSTR2 and HA-SSTR2Δ314 are competent for imaging in vivo. FIG. 6A: Representative planar γ-camera image of a nude mouse demonstrates that tumors derived from HT1080 cells expressing HA-wt SSTR2 (left shoulder) or expressing HA-SSTR2Δ314 (right shoulder) are visible, but the tumor derived from HT 1080 cells transfected with vector is not visible (left thigh). FIG. 6B: Nude mice bearing subcutaneous tumors were injected intravenously with ^{l u}In-octreotide (13 MBq) and imaged 24 hours later. Greater in vivo biodistribution of the radiopharmaceutical is seen in tumors derived from HT 1080 cells expressing HA-wt SSTR2 or HA-SSTR2Δ314 cells than in tumors derived from cells transfected with vector. "%ID/g" refers to percent injected dose per gram. Error bars represent standard deviation (n = 6 mice; *P < 0.05).

FIG. 7. Stably transfected HS-5 cells express similar amounts of HA-SSTR2Δ314 (A314) or HA-wt SSTR2 (wt). Quantitative ELISA using an antibody to the HA-domain demonstrates equal binding. See legend to FIG. 1. Error bars represent SD of triplicate samples. "mU" refers to milliunits of horse radish peroxidase enzymatic activity.

FIG. 8. Stably transfected HS-5 cells expressing HA-SSTR2Δ314 (A314) or HA-wt SSTR2 (wt) bind similar amounts of ¹¹¹In octreotide. Binding was performed using 10^"7 M ¹¹¹In octreotide and competition was performed using 10^"6 M somatostatin. Together with the the previous ELISA of the HS5 cells, these results show that the cells have equal fusion protein expression (ELISA targeting the HA tag) and equal binding to H l-In octreotide implying that the receptor mutation does not inhibit the ability of the receptor to bind the imaging ligand 1 H-In octreotide. This conclusion is also reached by IBand of FIGS. 2 A-D.

FIG. 9. HA-SSTR2Δ314 is signaling deficient for the cAMP pathway in human bone marrow mesenchymal cells, HS-5. Upon ligand (100 nM somatostatin- 14) binding, HA-wt

SSTR2 decreases forskolin induced cAMP production (wt) whereas HA-SSTR2Δ314 (Δ314) does not when expressed in HS-5 cells (*P < 0.05). HS-5 cells were stably transfected with HA-wt SSTR2 or HA-SSTR2Δ314.

FIG. 10. HA-SSTR2Δ314 is signaling deficient for the cGMP pathway in human bone marrow mesenchymal cells, HS-5. Upon ligand (100 nM somatostatin- 14) binding, HA-wt SSTR2 (wt) incites cGMP production, whereas HA-SSTR2Δ314 (Δ314) does not when expressed in HS-5 cells (*P < 0.05).

FIG. 11. HA-SSTR2Δ314 is deficient in inhibiting cell growth in human bone marrow mesenchymal cells, HS-5. Upon ligand (100 nM Sandostatin) binding, HA-wt SSTR2 (wt) decreases proliferation induced by serum, whereas HA-SSTR2Δ314 (Δ314) does not when expressed in HS-5 cells (*P < 0.05). Values on the abscissa are expressed as percent of color product of cells treated with Sandostatin compared to the same cells not exposed to Sandostatin using a MTT assay.

FIG. 12. Human bone marrow mesenchymal cells, HS-5, expressing HA-SSTR2Δ314 differentiate into osteoclast lineage cells that produce calcium phosphate. Cells transfected with vector, HA-SSTR2Δ314 (Δ314), or HA-wt SSTR2 (wt) produced calcium phosphate upon exposure to osteogenic induction medium. In osteoclast differentiation medium, calcium phosphate production was equivalent in cells transfected with vector or HA- SSTR2Δ314, but decreased in cells transfected HA-wt SSTR2. (*P < 0.05).

FIGS. 13A-F. Human bone marrow mesenchymal cells, HS-5, expressing HA-SSTR2Δ314 differentiate into adipocyte lineage. Cells transfected with vector, as shown in FIGS. 13A andl3B, HA-wt SSTR2, as shown in FIGS. 13C and 13D, or HA-SSTR2Δ314, as shown in FIGS. 13E and 13F, demonstrate increased staining for fat using Oil Red O with exposure to adipogenic induction medium, as shown in FIGS. 13B, 13D, and 13F, compared to without exposure, as shown in FIGS. 13A, 13C, and 13E. Magnification 400X. FIG. 14. In vivo imaging of cell trafficking of human bone marrow mesenchymal cells, HS- 5. expressing HA-SSTR2Δ314. HS-5 cells stably transfected with (A) HA-SSTR2Δ314, (B) HA-wt SSTR2, or (C) vector were exposed to 10^"7 M 1 H-In octreotide for two hours, washed and then injected into C57/B16 mice via tail vein. (D) No cell control incubated with 10^"7 M H l-In octreotide for two hours, washed and then injected into C57/B16 mice via tail vein. Increased signal overlies the lungs (arrow) in mice injected with HS-5 cells expressing (A) HA-SSTR2Δ314 or (B) HA-wt SSTR2HS-5. The planar gamma camera image was collected from 10 to 60 minutes post injection. FIG. 15. SSTR2Δ314 is signaling deficient for the cAMP pathway in human bone marrow mesenchymal cells, H S -5. Upon ligand (100 nM somatostatin- 14) binding, wild type SSTR2 decreases forskolin-induced cAMP production (wt) whereas SSTR2Δ314 (Δ314) does not when expressed in HS-5 cells (*P<0.05, Forskolin vs. Fors+SS). HS-5 cells were stably transfected with wt SSTR2 or SSTR2Δ314.

FIG. 16. SSTR2Δ314 is signaling deficient for the cGMP pathway in human bone marrow mesenchymal cells, HS-5. Upon ligand (100 nm somatostatin- 14) binding, wt SSTR2 (wt) incites cGMP production, whereas SSTR2Δ314 (Δ314) does not when expressed in HS-5 cells (*P<0.05, PBS vs. SS 14). FIG. 17. SSTR2Δ314 is deficient in inhibiting cell growth in human bone marrow mesenchymal cells, HS-5. Upon ligand (100 nM Sandostatin) binding, wt SSTR2 (wt) decreases proliferation induced by serum, whereas SSTR2Δ314 (Δ314) does not when expressed in HS-5 cells (*P<0.05). Values on the abscissa are expressed as percent of color product of cells treated with Sandostatin compared to the same cells not exposed to Sandostatin using a MTT assay.

FIG. 18. Human bone marrow mesenchymal cells, HS-5, expressing SSTR2Δ314 differentiate into osteoclast lineage cells that produce calcium phosphate. Cells transfected with vector, SSTR2Δ314 (Δ314), or wt SSTR2 (wt) produced calcium phosphate upon exposure to osteogenic induction medium. In osteoclast differentiation medium, calcium phosphate production was equivalent in cells transfected with vector or SSTR2Δ314, but decreased in cells transfected with wt SSTR2. (*P < 0.05, vector vs. wt SSTR2).

FIG. 19. Human bone marrow mesenchymal cells, HS-5, expressing SSTR2Δ314 differentiate into adipocyte lineage. Cells transfected with vector (upper left, upper right), wt SSTR2 (middle left, middle right), or SSTR2Δ314 (lower left, lower right) demonstrate increased staining for fat using Oil Red O with exposure to adipogenic induction medium (upper right, middle right, lower right) compared to without exposure (upper left, middle left, lower left). Magnification 400 X.

FIG. 20A-C. In vivo imaging of mesenchymal stem cells, HS5 cells expressing HA-SSTR2 or HA-SSTR2Δ314 (FIG. 20A) or SSTR2 or SSTR2Δ314 (FIG. 20B), coiniected with ovarian cancer cells to differentiate and serve as stromal support for the ovarian tumor. Expression of either HA-SSTR2 (short arrow) or HA- SSTR2Δ314 (long arrow) is visualized in FIG. 2OA or SSTR2 (short arrow) or SSTR2Δ314 (long arrow) is visualized in FIG. 2OB. Negative controls, HS5 cells transfected with vector (arrowhead), are not seen in FIG. 2OA or B. Stably transfected HS5 cells were coinjected with ovarian cancer cells (HeyA8) and after tumor formation, the mice were injected with 300 microCuries of H l-In octreotide via tail vein. Planar imaging using a gamma camera was performed the next day. (FIG. 20C) Biodistribution. Increased uptake is seen in tumors incorporating HS5 cells expressing HA- SSTR2, HA- SSTR2Δ314, SSTR2, or SSTR2Δ314 compared to tumors incorporating HS5 cells expressing vector (P<0.05). No statistically significant difference is seen among tumors incorporating HS5 cells expressing HA-SSTR2, HA- SSTR2Δ314, SSTR2 or SSTR2Δ314.

FIG. 21. Isolated human peripheral blood white blood cells. Human peripheral white blood cells isolated via a Ficoll gradient and stained with Hematoxylin demonstrating nuclei. Magnification 400X

FIG. 22. Isolated human peripheral blood white blood cells infected with adenovirus containing an insert for HA-SSTR2Δ314 (Δ314) or HA- wt SSTR2 (wf) express the fusion proteins. Quantitative ELISA using an antibody to the HA-domain. Expression of HA-wt SSTR2 (wt) was greater than that of HA-SSTR2Δ314 (Δ314) in these sets of cells used for the cell trafficking experiment. P < 0.05.

FIG. 23. In vivo imaging of cell trafficking of isolated human peripheral white blood cells expressing HA-SSTR2Δ314. White blood cells infected with adenovirus containing an insert for (A) HA-wt SSTR2, (B) HA-SSTR2Δ314, or (C) control were exposed to 10^"7 M H l-In octreotide for two hours, washed and then injected into nude mice via tail vein. (D) No cell control incubated with 10^"7 M 111-In octreotide for two hours, washed and then injected into nude mice via tail vein. Increased signal overlies the lungs (arrow) in mice injected with white blood cells infected with adenovirus containing an insert for (A) HA-SSTR2Δ314 or (B) HA-wt SSTR2. Expression of HA-wt SSTR2 was greater than that of HA-SSTR2Δ314 in these sets of cells used for the cell trafficking experiment (see FIG. 22) and this is reflected in the imaging. The planar gamma camera image was collected from 10 to 60 minutes post injection.

FIGS. 24A-C. Example constructs incorporating HA-SSTR2Δ314. (Introns need not be used, but can be helpful to increase expression in transgenic animals and their positions in the construct may be varied.) FIG. 24A: Examples of constructs using constitutive promoters such as CMV and human Ubiquitin. FIG. 24B: A functional and tissue selective promoter to assess for activation of the albumin promoter and as a marker for hepatocytes, since albumin expression is essentially restricted to hepatocytes. FIG. 24C: Example of an amplified functional and tissue selective promoter (miniCMV) for amplifying expression from the albumin promoter.

FIG. 25. Transgenic mice express HA-SSTR2Δ314 mRNA in multiple organs. Reverse transcriptase-polymerase chain reaction of RNA derived from transgenic mice (Ml and M2) or non-transgenic parental strain mouse (P). Primers for HA-SSTR2Δ314 demonstrate expression of HA-SSTRΔ314 in only transgenic mice. Primers for the ubiquitously expressed β-actin demonstrate loading of RNA. "MW marker" signifies a molecular weight marker. "Blank" signifies that no RNA was added to the reaction. Transgenic mice were created using a construct with an ubiquitin promoter for driving expression of HA-

SSTRΔ314.

FIG. 26. Transgenic mice express SSTR2Δ314 protein in multiple organs. Western blot of protein derived from transgenic mice (Ml and M2) or non-transgenic parental strain mouse (P) demonstrate expression of HA-SSTRΔ314 in only transgenic mice. Bone marrow was combined from two different transgenic mice (T) to obtain enough protein for the Western blot. Transgenic mice were created using a construct with an ubiquitin promoter for driving expression of HA-SSTR2. The primary antibody targeted the HA domain.

FIG. 27. Transgenic mice express HA-SSTR2Δ314 protein in the liver. Western blot of protein derived from transgenic mouse (ALB) or non-transgenic parental strain mouse (wt) demonstrate expression of HA-SSTR2Δ314 in the liver of the transgenic mice. Transgenic mice were created using a construct with a miniCMV-albumin promoter for driving expression of HA-SSTR2Δ314. The primary antibody targeted the HA domain.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

A new form of imaging or molecular imaging that has developed during the past decade involves the in vivo imaging of a reporter gene. Reporter gene technology was first applied to in situ imaging of tissue sections (reviewed in Blasberg et al, 2003). The present invention provides for methods of imaging a cell and/or its progeny that involve transferring into the cell an expression construct encoding a reporter that includes a truncated recombinant GPCR amino acid sequence. For example, the inventors have found that certain truncated recombinant somatostatin receptor amino acid sequences are signaling defective and/or have altered internalization. Further, the inventors have found that expression of a truncated recombinant somatostatin receptor in a stem cell has no significant effect on differentiation of the stem cell or on inhibition of cAMP and activation of cGMP production of the stem cell.

In certain aspects, the present invention allows for the imaging of cells that contain modified somatostatin receptors without causing cAMP and cGMP signaling, or otherwise affecting cellular functions such as growth suppression. Furthermore, the inventors have found that the truncation of the C-terminus of the somatostatin receptors serves to uncouple imaging from signal transduction. The signaling deficient somatostatin receptor type 2

(SSTR2) can function as a reporter of gene transfer, and expression can be imaged in vivo. It has been found that by using ^π indium-labeled octreotide and a C-terminus truncated SSTR2, in vivo SSTR2A imaging can be uncoupled from signal transduction.

A. Cells

The cells that are employed in the methods of the present invention can be any type of cell, such as a eukaryotic cell or a prokaryotic cell. In some embodiments, the cells are stem cells or immune cells, such as immune progenitor cells.

The term "stem cell" generally refers to any cells that have the ability to divide for indefinite periods of time and to give rise to specialized cells. The definition of "stem cell" includes, but is not limited to: a) totipotent cells such as an embryonic stem cell, an extraembryonic stem cell, a cloned stem cell, a parthenogenesis derived cell, a cell reprogrammed to possess totipotent properties, or a primordial germ cell; b) pluripotent cell such as a hematopoietic stem cell, an adipose derived stem cell, a mesenchymal stem cell, a cord blood stem cell, a placentally derived stem cell, an exfoliated tooth derived stem cells, a hair follicle stem cell or a neural stem cell; and c) a tissue specific progenitor cell such as a precursor cell for the neuronal, hepatic, nephrogenic, adipogenic, osteoblastic, osteoclastic, alveolar, cardiac, intestinal, or endothelial lineage.

The cells that are employed in the methods of the present invention can be obtained from any source known to those of ordinary skill in the art. In some embodiments, for example, the cells are stem cells obtained from a donor. In other embodiments, the cells are obtained from the subject who is to receive the cells as part of a therapeutic procedure. The cells can be derived, for example, from tissues such as pancreatic tissue, liver tissue, smooth muscle tissue, striated muscle tissue, cardiac muscle tissue, bone tissue, bone marrow tissue, bone spongy tissue, cartilage tissue, liver tissue, pancreas tissue, pancreatic ductal tissue, spleen tissue, thymus tissue, Peyer's patch tissue, lymph nodes tissue, thyroid tissue, epidermis tissue, dermis tissue, subcutaneous tissue, heart tissue, lung tissue, vascular tissue, endothelial tissue, blood cells, bladder tissue, kidney tissue, digestive tract tissue, esophagus tissue, stomach tissue, small intestine tissue, large intestine tissue, adipose tissue, uterus tissue, eye tissue, lung tissue, testicular tissue, ovarian tissue, prostate tissue, connective tissue, endocrine tissue, and mesentery tissue. In particular embodiments, the cells that are employed in the methods of the present invention are hematopoietic stem cells. The hematopoietic stem cells can be obtained, for example, from the blood or bone marrow of a subject. Further, stem cells of different tissue types (other than hematopoitic stem cells) can be obtained from the blood.

The stem cells to be expanded can be isolated from any organ of any mammalian organism, by any means known to one of skill in the art. The stem cells can be derived from embryonic or adult tissue. One of skill of the art can determine how to isolate the stem cells from the particular organ or tissue of interest, using methods known in the art. In a particular embodiment, the stem cells are isolated from same as prior paragraph. For example, the stem cells can be obtained from blood or bone marrow.

One of skill in the art will be able to determine a suitable growth medium for initial preparation of stem cells. Commonly used growth media for stem cells includes, but is not limited to, Iscove's modified Dulbecco's Media (IMDM) media, DMEM, KO-DMEM, DMEM/F12, RPMI 1640 medium, McCoy's 5 A medium, minimum essential medium alpha medium (.alpha.-MEM), F-12K nutrient mixture medium (Kaighn's modification, F-12K), X- vivo 20, Stemline, CClOO, H2000, Stemspan, MCDB 131 Medium, Basal Media Eagle (BME), Glasgow Minimum Essential Media, Modified Eagle Medium (MEM), Opti-MEM I Reduced Serum Media, Waymouth's MB 752/1 Media, Williams Media E, Medium NCTC- 109, neuroplasma medium, BGJb Medium, Brinster's BMOC-3 Medium, CMRL Medium, CO₂-Independent Medium, Leibovitz's L- 15 Media, and the like. If desired, other components, such as growth factors, can be added. Exemplary growth factors and other components include, but are not limited to, thrombopoietin (TPO), stem cell factor (SCF), IL-I, IL-3, IL-7, flt-3 ligand (fit-3L), G-CSF, GM-CSF, Epo, FGF-I, FGF-2, FGF-4, FGF-20, IGF, EGF, NGF, LIF, PDGF, bone morphogenic proteins (BMP), activin-A, VEGF, forskolin, glucocorticords, and the like. Furthermore, the media can contain either serum such as fetal calf, horse, or human serum, or more preferably, serum substitution components. Numerous agents have been introduced into media to alleviate the need for serum. For example, serum substitutes have included bovine serum albumin (BSA), insulin, 2-mercaptoethanol and transferrin (TF). One of ordinary skill in the art would be familiar with these supplementary components that can be added to the media.

The stem cells can then be stored for a desired period of time, if needed. Stem cell storage methods are well-known to those of skill in the art.

The stem cells can be sorted prior to administration by methods known in the art, using, for example, antibody technology such as fluorescence activated cell sorting (FACS), magnet activated cell sorting methods (e.g., magnetic resonance beads), column chromatography, or to isolate cells having the desired stem cell markers, or to remove unwanted, contaminating cell types having unwanted cell markers. For example, stem cells expressing a truncated recombinant GPCR amino acid sequence can be isolated from cells that do not expression a truncated recombinant GPCR amino acid sequence using any of these techniques.

The stem cells can have transferred an expression construct encoding a reporter comprising a GPCR or truncated recombinant GPCR prior to introduction of the cells to the subject at any stage in the preparation. In particular embodiments, the GPCR may be a somatostatin receptor, SSTR2A, a truncated somatostatin receptor, a truncated SSTR2A, SSTR2delta314. In some embodiments, the reporter encodes a protein tag fused to the N- terminal end or C-terminal end of the truncated GPCR amino acid sequence. The protein tag may or may not have enzymatic activity. In embodiments wherein the protein tag has enzymatic activity, the protein tag may be, for example, hemagglutinin A, beta-galactosidase, thymidine kinase, transferrin, myc-tag, VP 16, (His)₆-tag, FLAG, or chloramphenicol acetyl transferase.

B. Recombinant G-Protein Coupled Receptors (GPCRs)

GPCRs are a class of proteins involved in signal transduction, and are one of the largest receptor superfamilies known. These receptors are biologically important, and malfunction of these receptors has been shown to result in diseases such as Alzheimer disease, Parkinson disease, diabetes, dwarfism, color blindness, retinitis pigmentosa and asthma. GPCRs are also involved in depression, schizophrenia, sleeplessness, hypertension, anxiety, stress, renal failure and in several other cardiovascular, metabolic, neural, oncologic and immune disorders (Horn and Vriend, 1998). They have also been shown to play a role in HIV infection (Feng et al, 1996).

GPCRs have been characterized as having seven putative transmembrane domains that are connected by loops. The N-terminus is always extracellular and C-terminus is intracellular. The signal, such as an endogenous ligand or chemical moiety, is received at the extracellular N-terminus side. This signal is then transduced through the membrane to the cytosolic side where a heterotrimeric protein G-protein is activated which in turn elicits a response (see Horn and Vriend, 1998). GPCRs include a wide range of biologically active receptors, such as hormone receptors and neuronal receptors. Examples include, but are not limited to somatostatin receptors and adrenergic receptors.. Aspects of the invention include non-invasive imaging and/or therapy associated with the introduction of recombinant GPCRs into a cell of interest. Certain embodiments of the present invention generally pertain to nucleic acids encoding a recombinant GPCR amino acid sequences. The recombinant GPCR, for example, may be a recombinant GPCR. Exemplary GPCRs include the acetylcholine receptor: Ml, M2, M3, M4, or M5; adenosine receptor: Al; A2A; A2B; or A3; adrenoceptors: alphalA, alphalB, alphalD, alpha2A, alpha2B, alpha2C betal, beta2, or beta3; angiotensin receptors: ATI, or AT2; bombesin receptors: BBl, BB2, or BB3; bradykinin receptors: Bl, B2, calcitonin, Ainilin, CGRP, or adrenomedullin receptors; cannabinoid receptors: CBl, or CB2; chemokine receptors: CCRl, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCRlO, CXCRl, CXCR2, CXCR3, CXCR4, CXCR5, CX3CR1, or XCRl; chemotactic receptors : C3a, C5a, or fMLP; cholecystokinin and gastrin receptors: CCKl, or CCK2; corticotropin-releasing factor receptors: CRFl, or CRF2; dopamine receptors: Dl, D2, D3, D4, or D5; endothelin receptors: ET(A) or ET(B); galanin receptors: GALl, GAL2, or GAL3; glutamate receptors: mgll, mgl2, mgl3, mgl4, mgl5, mgl6, mgl7, or mgl8; glycoprotein hormone receptors: FSH, LSH, or TSH; histamine receptors: Hl, H2, H3, or H4; 5-HT receptors: 5-HT1A, 5-HT1B, 5- HTlD, 5-HT1B, 5-HT1F, 5HT2A, 5-HT2F, 5-HT2C, 5-HT3, 5-HT4, 5-HT5A, 5-HT5B, 5- HT6, or 5-HT7; leukotriene receptors: BLT, CysLTl, or CysLT2; lysophospholipid receptors: edgl, edg2, edg3, or edg4; melanocorlin receptors: MCl; MC2; MC3; MC4, or MC5; melatonin receptors: MTl, MT2, or MT3; neuropeptide Y receptors: Yl, Y2, Y4, Y5, or Y6; neurotension receptors: NTSl, or NTS2; opioids: DOP, KOP, MOP, or NOP; P2Y receptors: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, or P2Y12); peroxisome proliferators: PPAR- alpha, PPAR-beta, or PPAR-gamma; prostanoid receptors: DP, FP, IP, TP, EPl, EP2, EP3, or EP4; protease-activated receptors: PARl, PAR2, PAR3, or PAR4; Somatostatin receptors: SSTRl, SSTR2, SSTR2A, SSTR3, SSTR4, or SSTR5; tachykinin receptors: NKl, NK2, or NK3; thyrotropin-releasing hormone receptors: TRHl, or TRH2; urotensin-II receptor; vasoactivate intestinal peptide or pituitary adenylate cyclase activating peptide receptors: VPACl, VPAC2, or PACl; or vasopressin or oxytocin receptors: Via, VIb, V2, or OT.

In certain embodiments, the GPCR is a somatostatin receptor, such as a somatostatin type 2 receptor. Information pertaining to somatostatin receptors can be found in U.S. Patent Application Pub. No. 2002/0173626, which is herein specifically incorporated by reference in its entirety. Additional information regarding sequences of somatostatin receptors is set forth in Table 1 :

Table 1: Sequence Summary

*GenBank Accession Number pertains to nucleic acid sequence and encoded protein. Detailed information regarding the splice variants of human SSTR2 and its genomic structure can be found in Petersenn et al., 1999, herein specifically incorporated by reference. Additional information regarding the sequence of SSTR2 can be found in Yamada et al. (1992) and Vanetti et al. (1992). The nucleic acid encoding the GPCR amino acid sequence may encode an entire

GPCR sequence, a functional GPCR protein domain, a stably expressed non-functional GPCR, a GPCR polypeptide, or a GPCR polypeptide equivalent, each of which may include one or more transmembrane, extracellular, intracellular, extracelullar loop(s) and/or intracellular loop(s). The nucleic acids may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism, mRNA from a particular organism, and/or synthesized by use of various methods including but not limited to PCR™.

In some embodiments, the nucleic acid may be complementary DNA (cDNA). cDNA is DNA prepared using messenger RNA (mRNA) as a template. Thus, a cDNA does not contain any interrupted coding sequences and usually contains almost exclusively the coding region(s) for the corresponding protein. In other embodiments, the nucleic acid may be produced synthetically.

It may be advantageous to combine portions of the genomic DNA with cDNA or synthetic sequences to generate specific constructs. For example, where an intron is desired in the ultimate construct, a genomic clone may need to be used. Introns may be derived from other genes in addition to GPCR. The cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.

The present invention also includes nucleic acids encoding truncated GPCR polypeptide equivalents or other reporter polypeptide equivalents. These nucleic acids encoding reporter or GPCR polypeptide equivalents may be naturally-occurring homologous nucleic acid sequences from other organisms. A person of ordinary skill in the art would understand that commonly available experimental techniques can be used to identify or synthesize nucleic acids encoding reporter or GPCR polypeptide equivalents. The present invention also encompasses chemically synthesized mutants of these sequences. Another kind of sequence variant results from codon variation. Because there are several codons for most of the 20 normal amino acids, many different DNAs can encode GPCRs. The codons include: Alanine (Ala): GCA, GCC, GCG, and GCU; Cysteine (Cys): UGC and UGU; Aspartic acid (Asp): GAC and GAU; Glutamic acid (GIu): GAA and GAG; Phenylalanine (Phe): UUC and UUU; Glycine (GIy): GGA, GGC, GGG and GGU; Histidine (His): CAC and CAU; Isoleucine (He): AUA, AUC and AUU; Lysine (Lys): AAA and AAG; Leucine (Leu): UUA, UUG, CUA, CUC, CUG and CUU; Methionine (Met): AUG; Asparagine (Asn): AAC and AAU; Proline (Pro): CCA, CCC, CCG and CCU; Glutamine (GIn): CAA and CAG; Arginine (Arg): AGA, AGG, CGA, CGC, CGG and CGU; Serine (Ser): AGC, AGU, UCA, UCC, UCG and UCU; Threonine (Thr): ACA, ACC, ACG and ACU; Valine (VaI): GUA, GUC, GUG and GUU; Tryptophan (Trp) UGG; Tyrosine (Tyr): UAC and UAU. As stated above, the GPCR encoding sequences and other reporter sequences set forth herein may be full length genomic or cDNA copies, or fragments thereof. The present invention also may employ shorter oligonucleotides of the reporters or GPCRs. Sequences of 12 bases long should occur only once in the human genome and, therefore, suffice to specify a unique target sequence or PCR oligo. Both binding affinity and sequence specificity of an oligonucleotide to its complementary target increases with increasing length. It is contemplated that oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or longer base pairs will be used, for example, in the preparation of GPCR mutants and in PCR reactions.

1. Somatostatin Receptors In particular embodiments of the present invention, the truncated recombinant GPCR amino acid sequence is a truncated recombinant somatostatin receptor amino acid sequence. There are six somatostatin receptors (SSTR), types 1, 2A and 2B, 3, 4, and 5. Types 2A and 2B are alternate splice variants that are identical, except that type 2A has a longer intracytoplasmic C-terminus. Human type 2 has the highest affinity for the FDA approved somatostatin analogue, ¹¹¹In labeled octreotide. This radiopharmaceutical, approved for whole body imaging, and 99mTc labeled analogs, approved for lung imaging, are used in clinical practice to detect tumors over-expressing somatostatin receptors, such as neuroendocrine tumors. The normal biodistribution and dosimetry of radiolabeled somatostatin analogs used for imaging clinically has been well studied. The radiopharmaceutical is normally found in the kidneys, bladder, liver, spleen and bowel after intravenous injection. At the tracer doses used for imaging, no side-effects greater than placebo are found and patients are routinely imaged serially. Clinically, increased SSTR2 expression renders even small tumors detectable. PET based agents are also being developed.

For this receptor, in vitro studies suggest that the sixth and seventh transmembrane domains are essential for binding octreotide. Transmembrane domains three through five may also be important because a cysteine-cysteine disulfide bond is predicted between transmembrane domains three and extracellular domain two. Transmembrane domains three through seven have been predicted to cooperate in forming the pocket for binding octreotide.

SSTR2 regulates cAMP production. Gambhir et al. (1999) found that a D2 receptor mutant deficient in regulating cAMP can still be imaged. No functional (phenotypic changes) cellular changes were assessed such as effects on proliferation. In COS-7 cells, activation of human SSTR2 results in decreased cAMP production and activation of phospholipase C and calcium mobilization fully or partially, respectively, via a pertussis toxin sensitive G-protein. Through cAMP, somatostatin can regulate secretion. In 32D hematopoietic cells, cAMP appears to be required for SSTR2 mediated chemotaxis. The cytoplasmic C-terminus of the somatostatin receptor is involved in regulating cAMP. Deletion of amino acids beyond 349 of rat SSTR2 increases basal cAMP inhibition in human embryonic kidney (HEK 293) cells. Deletion of amino acids beyond 318 of human SSTR5 eliminates inhibition of cAMP in Chinese hamster ovary (CHO Kl) cells.

Inhibition of proliferation by SSTR2 involves multiple downstream mediators including phosphatases. The tyrosine phosphatase SHP-I is regulated by SSTR2, but SHP-I does not appear to regulate cAMP in the breast carcinoma line MCF-7. Upstream of SHP-I are reported to be inhibitory G proteins, the tyrosine phosphatase SHP-2 and the tyrosine kinase Src. SHP-2 interacts with SSTR2 tyrosine 228 in the context LCYLFI in the third intracellular domain and tyrosine 312 in the context of ILYAFL in transmembrane domain 7 next to the C-terminus. How Src associates with the SSTR2 has not yet been clarified. The phosphatases may have direct effect on phosphorylation of the somatostatin receptor itself, stimulatory growth factors or other downstream effectors. Phosphatidyl inositol, Ras, Rapl,

B-raf, MEKl and 2, Map kinase/Erk 1 and 2 have been implicated in SSTR2 mediated signaling in CHO DG44 cells; but in neuroblastoma cells, Ras did not appear to be involved and Map kinase/Erk 1 and 2 activity decreased, instead of increased as in CHO DG44 cells.

Thus, the role of the MAP kinase pathway in mediating inhibition of proliferation by SSTR2 is not yet clear. Also downstream of SHP-I is the neuronal nitric oxide synthase (nNOS) and guanylate cyclase, both of which appear necessary for SSTR2 mediated inhibition of proliferation in CHO cells and mouse pancreatic acinar cells. The inhibition may also involve other phosphotyrosine phosphatases and more downstream effectors such as cyclin dependent kinase inhibitor p27kipl. Somatostatin also regulates transcription factors such as c-jun, c-fos and AP-I. 2. Truncated GPCRs

For signaling, the C-terminus and intracytoplasmic domains of SSTR2 appear to be involved. As stated above, for both rat SSTR2 and human SSTR5, deletion analysis has demonstrated that the cytoplasmic C-terminus regulates inhibition of cAMP production. In particular, deletion of the SSTR2 after amino acid 314 is signaling defective and can be imaged in vivo. Truncation can be at either the N-terminus or the C-terminus or both termini.

C. Nucleic Acids, Expression Constructs, and Promoters

Aspects of the invention include transfecting a cell with an expression construct comprising a first region that is a nucleic acid sequence encoding a first reporter comprising a truncated recombinant GPCR amino acid sequence operatively linked to a first promoter sequence. The GPCR may be a recombinant GPCR that produces or binds to or enzymatically acts upon agents that produce a detectable signal. In other aspects, expression construct may include one or more additional nucleic acid sequences, such as additional reporters, additional coding regions, or additional promoters

The term "nucleic acid" is well known in the art. A "nucleic acid" as used herein will generally refer to a molecule (i.e., a strand) of DNA, RNA or a derivative or analog thereof, comprising a nucleobase. A nucleobase includes, for example, a naturally occurring or derivatized purine or pyrimidine base found in DNA (e.g., an adenine "A," a guanine "G," a thymine "T" or a cytosine "C") or RNA (e.g., an A, a G, an uracil "U" or a C). The term "nucleic acid" encompass the terms "oligonucleotide" and "polynucleotide," each as a subgenus of the term "nucleic acid." The term "oligonucleotide" refers to a molecule of between about 3 and about 100 nucleobases in length. The term "polynucleotide" refers to at least one molecule of greater than about 100 nucleobases in length.

These definitions generally refer to a single-stranded molecule, but in specific embodiments will also encompass an additional strand that is partially, substantially or fully complementary to the single-stranded molecule. Thus, a nucleic acid may encompass a double-stranded molecule or a triple-stranded molecule that comprises one or more complementary strand(s) or "complement(s)" of a particular sequence comprising a molecule The term "vector" is used to refer to a carrier into which a nucleic acid sequence can be inserted for introduction into a cell where it can be expressed and/or replicated. The term "expression vector" or "nucleic acid vector" refers to a nucleic acid containing a nucleic acid sequence or "cassette" coding for at least part of a nucleic acid sequence, also referred to herein as a gene, product capable of being transcribed and "regulatory" or "control" sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that govern transcription and translation, the expression vectors may contain nucleic acid sequences that serve other functions as well. 1. Promoters

The term "promoter" is used interchangeably with "promoter element" and "promoter sequence." Likewise, the term "enhancer" is used interchangeably with "enhancer element" and "enhancer sequence." A promoter, enhancer, or repressor, is said to be "operably linked" to a nucleic acid or transgene, such as a nucleic acid encoding a recombinant seven transmembrane G-protein associated receptor, when such element(s) control(s) or affect(s) nucleic acid or transgene transcription rate or efficiency. For example, a promoter sequence located proximally to the 5' end of a transgene coding sequence is usually operably linked with the transgene. As used herein, term "regulatory elements" is used interchangeably with "regulatory sequences" and refers to promoters, enhancers, polyadenylation sites and other expression control elements, or any combination of such elements.

Promoters are positioned 5' (upstream) to the genes that they control. Many eukaryotic promoters contain two types of recognition sequences: TATA box and the upstream promoter elements. The TATA box, located 25-30 bp upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase II to begin RNA synthesis at the correct site. In contrast, the upstream promoter elements determine the rate at which transcription is initiated. These elements can act regardless of their orientation, but they must be located within 100 to 200 bp upstream of the TATA box.

Enhancer elements can stimulate transcription up to 1000-fold from linked homologous or heterologous promoters. Enhancer elements often remain active even if their orientation is reversed (Li et ah, 1990). Furthermore, unlike promoter elements, enhancers can be active when placed downstream from the transcription initiation site, e.g., within an intron, or even at a considerable distance from the promoter (Yutzey et ah, 1989).

As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the positioning of regulatory elements with respect to the transgene may vary significantly without loss of function. Multiple copies of regulatory elements can act in concert. Typically, an expression vector comprises one or more enhancer sequences followed by, in the 5' to 3' direction, a promoter sequence, all operably linked to a transgene followed by a polyadenylation sequence. A "promoter" sequence is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrases "operatively positioned," "operatively linked," "under control," and "under transcriptional control" mean that a promoter is in a correct functional location and orientation in relation to a nucleic acid sequence to control transcriptional initiation and expression of that sequence. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence. Together, an appropriate promoter or promoter/enhancer combination, and a gene of interest, comprise an expression cassette. One or more expression cassettes may be present in a given nucleic acid vector or expression vector. In certain aspects, one expression cassette may encode a transactivator that interacts with a promoter of a second expression cassette. The one or more expression cassettes may be present on the same and/or different expression vector.

A promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating a portion the 5' non-coding sequences located upstream of the coding segment or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. In certain aspect of the invention a heterologous promoter may be a chimeric promoter, where elements of two or more endogenous, heterologous or synthetic promoter sequences are operatively coupled to produce a recombinant promoter. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (see U.S. Patents 4,683,202 and 5,928,906, each incorporated herein by reference). Such promoters may be used to drive reporter expression, which include, but are not limited to GPCRs, β-galactosidase or luciferase to name a few. Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

A promoter and/or enhancer will typically be used that effectively directs the expression of the DNA segment in a cell type, organelle, and organism chosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (2001), incorporated herein by reference. The promoters employed may be constitutive, tissue- selective, inducible, lineage-specific, or function-specific and/or useful under the appropriate conditions to direct expression of the introduced DNA segment, such as is advantageous in the production of proteins, recombinant proteins and/or peptides. The promoter may be heterologous or endogenous or a combination thereof. The position of the promoter/ may be varied. It is contemplated that 1, 2, 3, 4, or more expression cassettes may be present in a particular vector or a particular cell with no general preference as to the order of the cassettes in an expression vector. A first, second, third or fourth promoter of an expression cassette may be a constitutive, tissue selective, lineage specific, or function-specific promoter sequence that drives expression of a gene of interest, such as a reporter, a signaling sequence, a trafficking sequence, or a therapeutic gene.

Certain aspects of the invention include promoter sequences that interact with endogenous or exogenous transactivators. In certain aspects a transactivator is a recombinant transactivator. A recombinant transactivator may be expressed in cells into which a nucleic acid of the invention is introduced. Alternatively, a recombinant transactivator or a nucleic acid encoding a recombinant transactivator may be introduced before, with or after a nucleic acid of the invention. In certain aspects, the recombinant transactivator may be encoded in a nucleic acid encoding an imaging or therapeutic agent. A promoter may be functional in a variety of tissue types and in several different species of organisms, or its function may be restricted to a particular species and/or a particular normal or diseased tissue or cell type. Further, a promoter may be constitutively active, or it may be selectively activated by certain substances (e.g., a tissue-selective factor), under certain conditions (e.g., hypoxia, or the presence of an enhancer element in the expression cassette containing the promoter), or during certain developmental stages of the organism (e.g., active in fetus, silent in adult). A "function-specific ppromoter sequence" is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled, wherein the sequence is active in cells and whose products perform a particular function of interest. Examples include insulin, T-cell receptor, immunoglobulin, hormone or paracrine promoters such as vascular endothelial growth factor, structural protein promoters such as dystrophin, intracellular components such as fat or melanin, or extracellular components such as cartilage.

Promoters useful in the practice of the present invention may be tissue-specific— that is, they are capable of driving transcription of a gene in one or a few normal or diseased tissue(s) while remaining largely "silent" or expressed at relatively low levels in other tissue types. It will be understood, however, that tissue-specific or tissue-selective promoters may have a detectable amount of "background" or "base" activity in those tissues where they are silent. The degree to which a promoter is selectively activated in a target tissue can be expressed as a selectivity ratio (activity in a target tissue/activity in a control tissue). In this regard, a tissue specific promoter useful in the practice of the present invention typically has a selectivity ratio of greater than about 1 :1.01, 1 :1.1, 1 :1.5, 1 :2, 1 :3, 1 :4, 1 :5 or more. Preferably, the selectivity ratio is greater than about 1 :1.5. The promoter may also function in a reverse manner with decreased activity in the normal or diseased tissue(s) of interest. It will be further understood that certain promoters, while not restricted in activity to a single tissue type, may nevertheless show selectivity in that they may be active in one group of tissues, and less active or silent in another group. Such promoters are also termed "tissue specific" or "tissue selective," and are contemplated for use with the present invention. For example, promoters that are active in a particular type of tissue may be therapeutically useful in diseases affecting the tissue that may be amenable to stem cell therapy.

The level of expression of a coding region under the control of a particular promoter can be modulated by manipulating the promoter region. For example, different domains within a promoter region may possess different gene-regulatory activities. The roles of these different regions are typically assessed using vector constructs having different variants of the promoter with specific regions deleted (i.e., deletion analysis) or base pair(s) mutated. Vectors used for such experiments typically contain a reporter sequence, which is used to determine the activity of each promoter variant under different conditions. Application of such a deletion analysis enables the identification of promoter sequences containing desirable activities and thus identifying a particular promoter domain, including core promoter elements, those elements when deleted detrimentally effect characteristics of the promoter, such as but not limited to selectivity or transcription factor binding. This approach may be used to identify, for example, the smallest region capable of conferring tissue specificity, or the smallest region conferring a robust transcriptional response when combined with other promoter elements, such as but not limited to the core CMV promoter or a mini-CMV.

A number of promoters, described herein, may be particularly advantageous in practicing the present invention. In most instances, these promoters may be isolated as convenient restriction digest fragments suitable for cloning into a selected vector. Alternatively, promoter fragments may be isolated using the polymerase chain reaction or by oligonucleotide synthesis. Cloning of these promoter fragments may be facilitated by incorporating restriction sites at the 5' ends of the primers.

One of ordinary skill in the art would be familiar with the various types of promoter sequences that can be employed in the context of the present invention. For example, an exemplary list of such promoters/enhancers includes, but is not limited to Immunoglobulin Heavy Chain (Banerji et al, 1983; Gilles et al, 1983; Grosschedl et al, 1985; Atchison et al, 1986, 1987; Imler et al, 1987; Weinberger et al, 1984; Kiledjian et al, 1988; Porton et al; 1990); Immunoglobulin Light Chain (Queen et al, 1983; Picard et al, 1984); T-CeIl Receptor (Luria et al, 1987; Winoto et al, 1989; Redondo et al; 1990); HLA DQ α and/or DQ β (Sullivan et al, 1987); β-Interferon (Goodbourn et al, 1986; Fujita et al, 1987; Goodbourn et al, 1988); Interleukin-2 (Greene et al, 1989); Interleukin-2 Receptor (Greene et al, 1989; Lin et al, 1990); MHC Class II 5 (Koch et al, 1989); MHC Class II HLA-Dra (Sherman et al, 1989); β-Actin (Kawamoto et al, 1988; Ng et al; 1989); Muscle Creatine Kinase (MCK) (Jaynes et al, 1988; Horlick et al, 1989; Johnson et al, 1989); Prealbumin (Transthyretin) (Costa et al, 1988; Elastase I, Ornitz et al, 1987); Metallothionein (MTII) (Karin et al, 1987; Culotta et al, 1989); Collagenase (Pinkert et al, 1987; Angel et al, 1987); Albumin (Pinkert et al, 1987; Tranche et al, 1989, 1990); α-Fetoprotein (Godbout et al, 1988; Campere et al, 1989; γ-Globin, Bodine et al, 1987; Perez-Stable et al, 1990); β- Globin (Trudel et al, 1987); c-fos (Cohen et al, 1987); c-UA-ras (Treisman, 1986; Deschamps et al, 1985); Insulin (Edlund et al, 1985); Neural Cell Adhesion Molecule (NCAM) (Hirsch et al, 1990); (X₁ -Antitrypsin (Latimer et al, 1990); H2B (TH2B) Histone (Hwang et al, 1990); Mouse and/or Type I Collagen (Ripe et al, 1989); Glucose-Regulated Proteins (GRP94 and GRP78) (Chang et al, 1989); Rat Growth Hormone (Larsen et al, 1986); Human Serum Amyloid A (SAA) (Edbrooke et al, 1989); Troponin I (TN I) (Yutzey et al, 1989); Platelet-Derived Growth Factor (PDGF) (Pech et al, 1989); Duchenne Muscular Dystrophy (Klamut et al, 1990); SV40 (Banerji et al, 1981; Moreau et al, 1981; Sleigh et al, 1985; Firak et al, 1986; Herr et al, 1986; Imbra et al, 1986; Kadesch et al, 1986; Wang et al, 1986; Ondek et al, 1987; Kuhl et al, 1987; Schaffher et al, 1988); Polyoma (Swartzendruber et al, 1975; Vasseur et al, 1980; Katinka et al, 1980, 1981; Tyndall et al, 1981; Dandolo et al, 1983; de Villiers et al, 1984; Hen et al, 1986; Satake et al, 1988; Campbell and Villarreal, 1988); Retroviruses (Kriegler et al, 1982, 1983; Levinson et al, 1982; Kriegler et al, 1983, 1984a, b, 1988; Bosze et al, 1986; Miksicek et al, 1986; Celander et al, 1987, 1988; Thiesen et al, 1988; Choi et al, 1988; Reisman et al, 1989); Papilloma Virus (Campo et al, 1983; Lusky et al, 1983; Spandidos and Wilkie, 1983; Spalholz et al, 1985; Lusky et al, 1986; Cripe et al, 1987; Gloss et al, 1987; Hirochika et al, 1987; Stephens et al, 1987); Hepatitis B Virus (Bulla et al, 1986; Jameel et al, 1986; Shaul et al, 1987; Spandau et al, 1988; Vannice et al, 1988); Human Immunodeficiency Virus (Muesing et al, 1987; Hauber et al, 1988; Jakobovits et al, 1988; Feng et al, 1988; Takebe et al, 1988; Rosen et al, 1988; Berkhout et al, 1989; Laspia et al, 1989; Sharp et al, 1989; Braddock et al, 1989); Cytomegalovirus (CMV) (Weber et al, 1984; Boshart et al, 1985; Foecking et al, 1986); and/or Gibbon Ape Leukemia Virus (Holbrook et al, 1987; Quinn et al, 1989).

2. Internal Ribosome Entry Sites (IRES) In certain embodiments of the invention, internal ribosome entry site (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991) and further sequences as well as modified versions are envisioned in this application for invention. IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (U.S. Patents 5,925,565 and 5,935,819; and PCT application PCT/US99/05781) and are envisioned in this application for invention. The order (upstream or downstream of the IRES) of the reporter and gene(s) of interest is not important for the invention. More than one gene of interest may be linked.

3. Selectable Markers

In certain embodiments of the invention, a nucleic acid construct of the present invention may be isolated or selected for in vitro or in vivo by including a selectable marker in the expression vector. Such selectable markers would confer an identifiable characteristic to the cell permitting easy identification, isolation and/or selection of cells containing the expression vector. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drug resistance marker. Examples of selectable and screenable markers are well known to one of skill in the art.

4. Other Elements of Expression Cassettes

Most transcribed eukaryotic RNA molecules will undergo RNA splicing to remove introns from the primary transcripts. Vectors containing genomic eukaryotic sequences may require donor and/or acceptor splicing sites to ensure proper processing of the transcript for protein expression (Chandler et ah, 1997).

One may include a polyadenylation signal in the expression construct to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and/or any such sequence may be employed. Specific embodiments include the SV40 polyadenylation signal and/or the bovine growth hormone polyadenylation signal, convenient and/or known to function well in various target cells. Also contemplated as an element of the expression cassette is a transcriptional termination site. These elements can serve to enhance message levels and/or to minimize read through from the cassette into other sequences. The vectors or constructs of the present invention may comprise at least one termination signal. A "termination signal" or "terminator" is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (polyA) to the 3' end of the transcript. RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently. Thus, in other embodiments involving eukaryotes, the terminator may comprise a signal for the cleavage of the RNA, and it is more specific that the terminator signal promotes polyadenylation of the message. The terminator and/or polyadenylation site elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

Terminators contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, the termination sequences of genes, such as for example the bovine growth hormone terminator or viral termination sequences, such as for example the SV40 terminator. In certain embodiments, the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation.

In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed "ori"), which is a specific nucleic acid sequence at which replication is initiated. Alternatively an autonomously replicating sequence (ARS) can be employed if the host cell is yeast.

D. Transfer of Expression Constructs

Aspects of the invention include transferring into a cell an expression construct comprising a nucleic acid sequence encoding a truncated recombinant GPCR operatively coupled to a promoter sequence. Techniques pertaining to the transfer of expression constructs into cells are well-known to those of ordinary skill in the art. Exemplary techniques are discussed below.

1. Viral Vectors

In certain embodiments of the present invention, transfer of an expression construct into a cell is accomplished using a viral vector. Techniques using "viral vectors" are well- known in the art. A viral vector is meant to include those constructs containing viral sequences sufficient to (a) support packaging of the expression cassette and (b) to ultimately express a recombinant gene construct that has been cloned therein. In particular embodiments, the viral vector is a lentivirus vector. Lentivirus vectors have been successfully used in infecting stem cells and providing long term expression.

Another method for delivery of a nucleic acid involves the use of an adenovirus vector. Adenovirus vectors are known to have a low capacity for integration into genomic DNA. Adenovirus vectors result in highly efficient gene transfer.

Adenoviruses are currently the most commonly used vector for gene transfer in clinical settings. Among the advantages of these viruses is that they are efficient at gene delivery to both nondividing and dividing cells and can be produced in large quantities. The vector comprises a genetically engineered form of adenovirus (Grunhaus et al, 1992). In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification.

Adenovirus is particularly suitable for use as a gene transfer vector because of its mid- sized genome, ease of manipulation, high titer, wide target-cell range and high infectivity. A person of ordinary skill in the art would be familiar with experimental methods using adenoviral vectors.

The adenovirus vector may be replication defective, or at least conditionally defective, and the nature of the adenovirus vector is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F and other serotypes or subgroups are envisioned. Adenovirus type 5 of subgroup C is the starting material in order to obtain the conditional replication- defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector. Adenovirus growth and manipulation is known to those of skill in the art, and exhibits broad host range in vitro and in vivo. Modified viruses, such as adenoviruses with alteration of the CAR domain, may also be used. Methods for enhancing delivery or evading an immune response, such as liposome encapsulation of the virus, are also envisioned. The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains two long terminal repeat (LTR) sequences present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding a nucleic acid or gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. A person of ordinary skill in the art would be familiar with well-known techniques that are available to construct a retroviral vector. Adeno-associated virus (AAV) is an attractive vector system for use in the present invention as it has a high frequency of integration and it can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells in tissue culture (Muzyczka, 1992). AAV has a broad host range for infectivity (Tratschin et al., 1984; Laughlin et al, 1986; Lebkowski et al, 1988; McLaughlin et al, 1988), which means it is applicable for use with the present invention. Details concerning the generation and use of rAAV vectors are described in U.S. Patents 5,139,941 and 4,797,368, each incorporated herein by reference.

Typically, recombinant AAV (rAAV) virus is made by cotransfecting a plasmid containing the gene of interest flanked by the two AAV terminal repeats (McLaughlin et al, 1988; Samulski et al, 1989; each incorporated herein by reference) and an expression plasmid containing the wild-type AAV coding sequences without the terminal repeats, for example pIM45 (McCarty et al., 1991; incorporated herein by reference). A person of ordinary skill in the art would be familiar with techniques available to generate vectors using AAV virus.

Herpes simplex virus (HSV) has generated considerable interest in treating nervous system disorders due to its tropism for neuronal cells, but this vector also can be exploited for other tissues given its wide host range. Another factor that makes HSV an attractive vector is the size and organization of the genome. Because HSV is large, incorporation of multiple genes or expression cassettes is less problematic than in other smaller viral systems. In addition, the availability of different viral control sequences with varying performance (temporal, strength, etc) makes it possible to control expression to a greater extent than in other systems. It also is an advantage that the virus has relatively few spliced messages, further easing genetic manipulations. HSV also is relatively easy to manipulate and can be grown to high titers. Thus, delivery is less of a problem, both in terms of volumes needed to attain sufficient MOI and in a lessened need for repeat dosings. For a review of HSV as a gene therapy vector, see

Glorioso et al. (1995). A person of ordinary skill in the art would be familiar with well- known techniques for use of HSV as vectors.

Vaccinia virus vectors have been used extensively because of the ease of their construction, relatively high levels of expression obtained, wide host range and large capacity for carrying DNA. Vaccinia contains a linear, double-stranded DNA genome of about 186 kb that exhibits a marked "A-T" preference. Inverted terminal repeats of about 10.5 kb flank the genome.

Other viral vectors may be employed as constructs in the present invention. For example, vectors derived from viruses such as poxvirus may be employed. A molecularly cloned strain of Venezuelan equine encephalitis (VEE) virus has been genetically refined as a replication competent vaccine vector for the expression of heterologous viral proteins (Davis et al., 1996). Studies have demonstrated that VEE infection stimulates potent CTL responses and it has been suggested that VEE may be an extremely useful vector for immunizations (Caley et al., 1997). It is contemplated in the present invention, that VEE virus may be useful in targeting dendritic cells.

A polynucleotide may be housed within a viral vector that has been engineered to express a specific binding ligand. The virus particle will thus bind specifically to the cognate receptors of the target cell and deliver the contents to the cell. A novel approach designed to allow specific targeting of retrovirus vectors was developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope.

This modification can permit the specific infection of hepatocytes via sialoglycoprotein receptors.

Another approach to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor were used. The antibodies were coupled via the biotin components by using streptavidin (Roux et al., 1989). Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus in vitro (Roux et al., 1989). 2. Nonviral Gene Transfer

Several non-viral methods for the transfer of nucleic acids into cells also are contemplated by the present invention. These include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al, 1990) DEAE- dextran (Gopal, 1985), electroporation (Tur-Kaspa et al, 1986; Potter et al, 1984), nucleofection (Trompeter et al, 2003), direct microinjection (Harland and Weintraub, 1985), DNA- loaded liposomes (Nicolau and Sene, 1982; Fraley et al, 1979) and lipofectamine- DNA complexes, polyamino acids, cell sonication (Fechheimer et al, 1987), gene bombardment using high velocity microprojectiles (Yang et al, 1990), polycations (Boussif et al, 1995) and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use. A person of ordinary skill in the art would be familiar with the techniques pertaining to use of nonviral vectors, and would understand that other types of nonviral vectors than those disclosed herein are contemplated by the present invention. In a further embodiment of the invention, the expression cassette may be entrapped in a liposome or lipid formulation. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. Also contemplated is a gene construct complexed with Lipofectamine (Gibco BRL). One of ordinary skill in the art would be familiar with techniques utilizing liposomes and lipid formulations.

E. Other Reporters

In certain embodiments of the present invention, the expression construct comprises a coding region that encodes a reporter other than a truncated recombinant GPCR. The term "reporter," "reporter gene" or "reporter sequence" as used herein refers to any genetic sequence or encoded polypeptide sequence that is detectable and distinguishable from other genetic sequences or encoded polypeptides present in cells. Preferably, the reporter sequence encodes a protein that is readily detectable either by its presence, its association with a detectable moiety or by its activity that results in the generation of a detectable signal. In particular, reporters that can be imaged non-invasively or with non-invasive techniques are envisioned.

In some embodiments, a reporter nucleic acid may encode a polypeptide having a tag. In association with this embodiment, the method may further comprise the step of contacting the host cell with a fluorescently labeled antibody specific for the tag, thereby labeling the host cell, which may be detected and/or isolated by FACS or other detection, sorting or isolation methods.

In various embodiments, a nucleic acid sequence of the invention comprises a reporter nucleic acid sequence or encodes a product that gives rise to a detectable polypeptide. A reporter is or encodes a reporter molecule which is capable of directly or indirectly generating a detectable signal. Generally, although not necessarily, the reporter gene includes a nucleic acid sequence and/or encodes a detectable polypeptide that is not otherwise produced by the cells. Many reporter genes have been described, and some are commercially available for the study of gene regulation (e.g., Alam and Cook, 1990, the disclosure of which is incorporated herein by reference). Signals that may be detected include, but are not limited to color, fluorescence, luminescence, isotopic or radioisotopic signals, cell surface tags, cell viability, relief of a cell nutritional requirement, cell growth and drug resistance. Reporter sequences include, but are not limited to, DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, membrane bound proteins including, for example, G-protein coupled receptors (GPCRs), somatostatin receptors, CD2, CD4, CD8, the influenza hemagglutinin protein, symporters (such as NIS) and others well known in the art, to which high affinity antibodies or ligands directed thereto exist or can be produced by conventional means, and fusion proteins comprising a membrane bound protein appropriately fused to an antigen tag domain from, among others, hemagglutinin or Myc.

In various embodiments, the desired level of expression of at least one of the reporter sequence is an increase, a decrease, or no change in the level of expression of the reporter sequence as compared to the basal transcription level of the reporter sequence. In a particular embodiment, the desired level of expression of one of the reporter sequences is an increase in the level of expression of the reporter sequence as compared to the basal transcription level of the reporter sequence.

In various embodiments, the reporter sequence encodes unique detectable proteins which can be analyzed independently, simultaneously, or independently and simultaneously. In certain embodiments, the reporter sequence encodes a protein that can be visualized non- invasively such as the SSTR2Δ314. In other embodiments, the host cell may be a eukaryotic cell or a prokaryotic cell. Exemplary eukaryotic cells include yeast and mammalian cells. Mammalian cells include human cells and various cells displaying a pathologic phenotype, such as cancer cells. F. Detectable Moieties In certain embodiments of the invention, a reporter, such as a truncated recombinant

GPCR amino acid sequence, may be imaged by detecting its association with a detectable moiety. A "detectable moiety" is defined herein to refer to any molecule that can attach, either directly or indirectly, to a reporter. Examples of detectable moieties are set forth above. For example, in some embodiments, the detectable moiety is a ligand. A ligand is defined herein to refer to an ion, a peptide, a oligonucleotide, aptamer, a molecule, a small molecule, or a molecular group that binds to another chemical entity or polypeptide to form a larger complex. In the context of the present invention, the ligand may bind to a reporter or to an amino acid sequence attached to the reporter sequence (e.g., such as a protein tag fused to the N-terminal end or C-terminal end of the reporter amino acid sequence) to form a larger complex. Any ligand known to those of ordinary skill in the art is contemplated for use as a ligand in the context of the present invention. In some embodiments of the present invention, a ligand may be contacted with the cell for imaging. The ligand may or may not be internalized by the cell. Where a reporter has become localized to the cell surface, the ligand, in these embodiments, may bind to or associate with the reporter. Any method of binding of the ligand to the reporter is contemplated by the present invention. In certain other embodiments, a ligand may become internalized by a cell. Once internalized the ligand may, but need not, bind to or associate with the reporter or a second reporter within the cell.

The detectable moiety may be a molecule or part of a molecule that has properties or is conjugated to a moiety such that it is capable of generating a signal that can be detected. Any imaging modality known to those of ordinary skill in the art can be applied to image a ligand. In some embodiments, the ligand is capable of binding to or being coupled to a molecule or part of a molecule that can be imaged. For example, the ligand may be capable of binding to or be coupled to a radionuclide, and the radionuclide can be imaged using nuclear medicine techniques known to those of ordinary skill in the art. For example, the ligand may be ^{l u}In-octreotide. Information regarding imaging using ^{l u}In-octreotide can be found in U.S. Patent App. Pub. No. 20020173626, herein specifically incorporated by reference. In other embodiments, for example, the ligand is capable of binding to or being coupled to a contrast agent that can be detected using imaging techniques well-known to those of ordinary skill in the art. For example, the ligand may be capable of binding to or being coupled to a CT contrast agent, an ultrasound agent, an optical agent, or an MRI contrast agent. In certain embodiments of the present invention, a detectable moiety can bind to the reporter, and the ligand in turn generates a signal that can be measured using an imaging modality known to those of ordinary skill in the art. In other embodiments, the ligand can bind to a protein tag that is fused to the reporter. Thus, for example, imaging would involve measuring a signal from the ligand, and this in turn would provide for localization of the reporter sequence within the cell or within a subject.

A variety of valent metal ions, or radionuclides, are known to be useful for radioimaging and can be employed as detectable moieties. Examples include, but are not limited to ⁶⁷Ga, ⁶⁸Ga, ""¹Tc, ¹¹¹In, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁶⁹Yb, ⁶⁰Cu, ⁶¹Cu, ⁶⁴Cu, ⁶²Cu, ²⁰¹Tl, ⁷²A, and ¹⁵⁷Gd. In certain embodiments of the present invention, the nucleic acid for use in the imaging methods of the present invention encodes an amino acid sequence that can be radiolabeled in vivo. Radiolabeling of the encoded reporter sequence can be direct, or it can be indirect, such as by radiolabeling of a ligand that can bind the protein tag or reporter sequence. Radiolabeled agents, compounds, and compositions provided by the present invention are provided having a suitable amount of radioactivity.

Once the encoded sequence is radiolabeled, it can be imaged for visualizing a site, such as a tumor in a mammalian body. In accordance with this invention, the radiolabel is administered by any method known to those of ordinary skill in the art. For example, administration may be in a single unit injectable dose, administered as a radiolabeled ligand. Any of the common carriers known to those with skill in the art, such as sterile saline solution or plasma, may be utilized. Generally, a unit dose to be administered has a radioactivity of about 0.01 mCi to about 300 mCi, preferably 5 mCi to about 30 mCi. The solution to be injected at unit dosage is usually from about 0.01 mL to about 10 mL.

After intravenous administration of the radiolabeled reagent, imaging of the organ or tumor in vivo can take place, if desired, in minutes, hours or even longer, after the radiolabeled reagent is introduced into a patient. In some instances, a sufficient amount of the administered dose will accumulate in the area to be imaged within about 0.1 of an hour. G. Imaging Techniques

Imaging of a detectable moiety may be performed using any method known to those of ordinary skill in the art. Examples include PET, SPECT, and gamma scintigraphy. In gamma scintigraphy, the radiolabel is a gamma-radiation emitting radionuclide and the radiotracer is located using a gamma-radiation detecting camera (this process is often referred to as gamma scintigraphy). The imaged site is detectable because the radiotracer is chosen either to localize at a pathological site (termed positive contrast) or, alternatively, the radiotracer is chosen specifically not to localize at such pathological sites (termed negative contrast). Some aspects of the present invention pertain to methods for tracking the location of a cell in a subject that involve detecting the location of the cell in the subject by contacting the cell with a detectably moiety that binds to the truncated recombinant GPCR that is expressed in the cell. Detection of the expressed GPCR amino acid sequence can be performed by any method known to those of ordinary skill in the art. For example, the reporter may be imaged by administration of a detctable moiety to a subject, wherein the detectable moiety is directed to the reporter amino acid sequence. In other embodiments, the detectable moiety is a radiolabeled probe, such as ^{l u}In-octreotide. In further embodiments, the detectable moiety is a probe that can be imaged optically, such as by fluorescence, near infrared, infrared, MR, or ultrasound. Any method known to those of ordinary skill in the art for measuring a signal derived from a reporter or an associated detectable moiety that attaches to the reporter is contemplated for inclusion in the present invention. Exemplary methods of detecting are as follows.

1. Gamma Camera Imaging

A variety of nuclear medicine techniques for imaging are known to those of ordinary skill in the art. Any of these techniques can be applied in the context of the imaging methods of the present invention to measure a signal from the reporter. For example, gamma camera imaging is contemplated as a method of imaging that can be utilized for measuring a signal derived from the reporter. One of ordinary skill in the art would be familiar with techniques for application of gamma camera imaging. In one embodiment, measuring a signal can involve use of gamma-camera imaging of an ¹¹¹In or ^99mTc conjugate, in particular ¹¹¹In- octreotide or ^99mTc-somatostatin analogue. Single photon emission tomography (SPECT) may also be performed for three dimensional localization. 2. Computerized Tomography (CT)

Computerized tomography (CT) is contemplated as an imaging modality in the context of the present invention. By taking a series of X-rays, sometimes more than a thousand, from various angles and then combining them with a computer, CT made it possible to build up a three-dimensional image of any part of the body. A computer is programmed to display two-dimensional slices from any angle and at any depth. The slices may be combined to build three-dimensional representations.

In CT, intravenous injection of a radiopaque contrast agent can assist in the identification and delineation of soft tissue masses when initial CT scans are not diagnostic. Similarly, contrast agents aid in assessing the vascularity of a soft tissue or bone lesion. For example, the use of contrast agents may aid the delineation of the relationship of a tumor and adjacent vascular structures.

CT contrast agents include, for example, iodinated contrast media. Examples of these agents include iothalamate, iohexol, diatrizoate, iopamidol, ethiodol, and iopanoate. Gadolinium agents have also been reported to be of use as a CT contrast agent (see, e.g.,

Henson et ah, 2004). For example, gadopentate agents has been used as a CT contrast agent

(discussed in Strunk and Schild, 2004).

3. Magnetic Resonance Imaging (MRI)

Magnetic resonance imaging (MRI) is an imaging modality that uses a high-strength magnet and radio-frequency signals to produce images. The most abundant molecular species in biological tissues is water. It is the quantum mechanical "spin" of the water proton nuclei that ultimately gives rise to the signal in imaging experiments and other nuclei can also be imaged. In MRI, the sample to be imaged is placed in a strong static magnetic field (1-12 Tesla) and the spins are excited with a pulse of radio frequency (RF) radiation to produce a net magnetization in the sample. Various magnetic field gradients and other RF pulses then act on the spins to code spatial information into the recorded signals. By collecting and analyzing these signals, it is possible to compute a three-dimensional image which, like a CT, SPECT, and PET image, is normally displayed in two-dimensional slices. The slices may be combined to build three-dimensional representations. Contrast agents used in MR or MR spectroscopy imaging differ from those used in other imaging techniques. Their purpose is to aid in distinguishing between tissue components with similar signal characteristics and to shorten the relaxation times (which will produce a stronger signal on Tl -weighted spin-echo MR images and a less intense signal on T2-weighted images). Examples of MRI contrast agents include gadolinium chelates, manganese chelates, chromium chelates, and iron particles.

4. PET and SPECT Imaging modalities that provide information pertaining to information at the cellular level, such as cellular viability, include positron emission tomography (PET) and single- photon emission computed tomography (SPECT). In PET, a patient ingests or is injected with a radioactive substance that emits positrons, which can be monitored as the substance moves through the body. Closely related to PET is single-photon emission computed tomography, or SPECT.

The major difference between the two is that instead of a positron-emitting substance, SPECT uses a radioactive tracer that emits high-energy photons. SPECT is valuable for diagnosing multiple illnesses including coronary artery disease, and already some 2.5 million SPECT heart studies are done in the United States each year. PET radiopharmaceuticals for imaging are commonly labeled with positron-emitters such as ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁸²Rb, ⁶²Cu, and ⁶⁸Ga. SPECT radiopharmaceuticals are commonly labeled with positron emitters such as ^99mTc, ²⁰¹Tl, and ⁶⁷Ga, ¹¹¹In. Important receptor- binding SPECT radiopharmaceuticals include [¹²³I]QNE, [¹²³I]IBZM, and [¹²³I]iomazenil. These tracers bind to specific receptors, and are of importance in the evaluation of receptor- related diseases

5. Optical Imaging

Optical imaging is another imaging modality that has gained widespread acceptance in particular areas of medicine. Examples include optical labeling of cellular components, and angiography such as fluorescein angiography and indocyanine green angiography of the eyes. Examples of optical imaging agents include, for example, fluorescein, a fluorescein derivative, indocyanine green, Oregon green, a derivative of Oregon green derivative, rhodamine green, a derivative of rhodamine green, an eosin, an erythrosin, Texas red, a derivative of Texas red, malachite green, nanogold sulfosuccinimidyl ester, cascade blue, a coumarin derivative, a naphthalene, a pyridyloxazole derivative, cascade yellow dye, dapoxyl dye. Optical imaging includes near infrared imaging and infrared imaging. Near infrared imaging has more tissue penetration and less background. 6. Ultrasound

Another biomedical imaging modality that has gained widespread acceptance is ultrasound. Ultrasound imaging has been used to provide realtime cross-sectional and even three-dimensional images of soft tissue structures and blood flow information in the body. High-frequency sound waves and a computer create images of blood vessels, tissues, and organs.

Ultrasound imaging of blood flow can be limited by a number of factors such as size and depth of the blood vessel. Ultrasonic contrast agents, a relatively recent development, include perfluorine and perfluorine analogs, which are designed to overcome these limitations by helping to enhance grey-scale images and Doppler signals.

7. Dual Imaging

In certain embodiments, imaging using more than one modality is performed. For example, as set forth above, the imaging modality may include, but are not limited to, CT, MRI, PET, SPECT, ultrasound, or optical imaging. Other examples of imaging modalities known to those of ordinary skill in the art are contemplated by the present invention.

The imaging modalities are performed at any time during or after administration of the composition comprising the diagnostically effective amount of the compound that comprises two imaging moieties. For example, the imaging studies may be performed during administration of the dual imaging compound of the present invention, or at any time thereafter. In some embodiments, the first imaging modality is performed beginning concurrently with the administration of the dual imaging agent, or about 1 sec, 1 hour, 1 day, or any longer period of time following administration of the dual imaging agent, or at any time in between any of these stated times. In some embodiments of the present invention a second imaging modality may be performed concurrently with the first imaging modality, or at any time following the first imaging modality. For example, the second imaging modality may be performed about 1 sec, about 1 hour, about 1 day, or any longer period of time following completion of the first imaging modality, or at any time in between any of these stated times. One of ordinary skill in the art would be familiar with performance of the various imaging modalities contemplated by the present invention. 8. Imaging of a Subject Following Stem Cell Administration

Imaging of a cell and/or its progeny can be performed following introduction of a cell into a subject. For example, imaging can be performed after about 1 second, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year, or any longer period of time following administration of the cell. In some embodiments, imaging and biodistribution analysis can be performed as described by Yang et ah, 2005. In other embodiments, imaging may be preformed after approximately one and one-half weeks. One of ordinary skill in the art would be familiar with generating a protocol to imaging cells, such as stem cells, following introduction of cells into a subject.

Imaging of a cell and/or its progeny that include an expressed truncated recombinant GPCR can be performed for several purposes. For example, imaging can be performed to follow the transit of cells, such as stem cells, in the body following introduction of the cells into a subject. Imaging can also be used to assess cell viability following introduction of the cells into a subject, and over the course of time. Further, imaging can also be performed to assess stem cell or immune cell localization in a subject. For example, placing the reporter under the control of a constitutive promoter would provide for constant expression that may be used to assess localization and viability of the cell. Imaging can be used to assess trans/differentiation or fusion. For example, placing the reporter under the control of a tissue-selective promoter sequence would provide for expression of a particular reporter only upon trans/differentiation or fusion of a cell or its progeny to a particular tissue/cell type.

Alternatively, imaging can be performed to assess an immune cell, stem cell or its progeny's expression from a promoter of a gene whose product performs a function of interest following introduction of the cell into a subject. For example, placing a reporter in the expression construct under the control of a function-specific promoter would provide for expression of the reporter in stem cells until trans/differentiation or fusion. Alternatively expression may occur upon differentiation of the cell into a cell capable of performing a specific function. As an example in lymphocytes, T-cell activation may be assessed using promoter elements that initiate transcription upon T-cell activation. Thus, imaging can be applied in a wide variety of contexts that are significant in the context of stem cell and immune cell therapy.

The reporter may be linked to a gene of interest, for example by an IRES or a bidirectional promoter, so that expression of the reporter may be used to track not only its own expression, but also that of the gene of interest. Examples of genes of interest include those whose products may function in homing, implantation or differentiation. With multiple promoter-reporter constructs transferred into a cell, combinations of the above may be evaluated including in vivo. For example, with multiple promoter-reporter constructs transferred individually or together, viability, localization, differentiation, functional expression, and indirect evaluation of expression of a linked gene of interest may be evaluated. When used in combination, different promoters and different reporters that can be identified either simultaneously or serially may need to be employed. Simultaneous evaluation may be performed for example if the reporter or its detectable moiety have separable characteristics, for example, different energies of emission of gamma rays that can be separated by a gamma camera. In certain embodiments, a combination of more than one imaging technique can be used to determine trafficking, viability, and/or differentiation of the cells. For example, MR and γ-camera imaging can be used to determine the biodistribution of radiopharmaceutical in tumors. Imaging can be performed following administration of a subject with a detectable moiety. Because the detectable moiety will have a limeited physical and biological life, imaging of the reporter can be performed repeatedly. In addition, cells that have been exposed to a detectable moiety can be introduced into the subject, and then the subject subjected to one or more imaging techniques following introduction of the cells. Imaging can be performed a single time or more than one time point following introduction of the cells into the subject allowing serial evaluation. Image acquisition can be performed by any method known to those of ordinary skill in the art.

The reporter within the cells can be imaged both in vivo and ex vivo. In certain embodiments, ex vivo imaging occurs on a biopsy sample of tissue obtained from the subject following introduction of the cell into the subject. As discussed above, in vzVo-imaging can also be performed using any of a variety of modalities known to those of ordinary skill in the art.

The reporter within the cells can be detected both in vivo and ex vivo. In certain embodiments, ex vivo evaluation occurs on a biopsy sample of tissue obtained from the subject following introduction of the cell into the subject. Ex vivo evaluation for the reporter can be performed using a variety of techniques including but not limited to autoradiography, immunologic techniques such as immunohistochemistry, ELISA or Western blotting, PCR™, optical, CT, MR, nuclear imaging, or ultrasound. As discussed above, in vzVo-imaging can also be performed using any of a variety of modalities known to those of ordinary skill in the art. H. Transgenic Animals

Other aspects of the present invention pertain to non-human transgenic animals whose genome includes a nucleic acid encoding a recombinant GPCR. In certain embodiments the GPCR is used for the purpose of non-invasive or invasive imaging. In particular embodiments, the GPCR is a somatostatin receptor. In particular embodiments, the somatostatin receptor is SSTR2A. In particular embodiments, the GPCR is truncated. In particular embodiments, the GPCR is truncated and inhibited in eliciting signaling and/or phenotypic change and/or internalization. In particular embodiments, the truncated GPCR is truncated a somatostatin receptor. In particular embodiments, the truncated somatostatin receptor is truncated SSTR2A. In particular embodiments, the truncated SSTR2A is SSTR2Δ314.

Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art, e.g. (U.S. Pat. No. 4,870,009; U.S. Pat. No. 4,736,866; U.S. Pat. No. 4,873,191). Other non-mice transgenic animals may be made by similar methods. A transgenic founder animal, which can be used to breed additional transgenic animals, can be identified based upon the presence of the transgene in its genome and/or expression of the transgene mRNA in tissues or cells of the animals. Transgenic animals can be bred to other transgenic animals carrying other transgenes. The term "non-human animals" is intended to include any vertebrate such as mammals, birds, reptiles, and amphibians. Suitable mammals include rodents {e.g., rat, mouse), non-human primates, sheep, dogs, cats, rabbits, and cows. Suitable birds include chickens, geese, and turkeys. "Transgenic animal" refers to non-naturally occurring non- human animal in which one or more of the cells of the animal contain heterologous nucleic acids encoding human a truncated recombinant GPCR, that has been introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.

Methods for generating transgenic animals of the present invention, including knockouts and knock-ins, are well known in the art. In particular embodiments, the animal is transgenic for truncated SSTR2 such as SSTR2AΔ314. The cells/tissues/organs from the transgenic animal may be studied in vitro or in vivo. This animal may be made to express the gene under the control of a constitutive, tissue specific, lineage specific, or functional promoter. Cells from the animal may be introduced into another animal not transgenic for the same reporter for studying cell trafficking, viability, engraftment, trans/differentiation or fusion, expression from a promoter of a gene of a function of interest, expression of a linked gene of interest, etc. as described elsewhere in the application. In certain embodiments, stem cells or immune cells are extracted from the transgenic animal and studied as described above after injection into another animal with or without pathology. In certain embodiments, immune cells are extracted from the transgenic animal and studied as described above after injection into another animal with or without pathology. The cells may be used after extraction or may be cultured and then introduced into another animal.

In another embodiment tissues such as islets and/or organs may be transplanted and the GPCR reporter may be used for example but not limited to track their viability, incorporation, and trafficking of cells that may migrate. For the transgenic animal, tissues such as islets and organs may be transplanted and the GPCR reporter may be used for example to track their viability, incorporation, and trafficking of cells that may migrate from the graft. Within this, stem cells expressing the GPCR reporter may be made to differentiate into tissues or organs {e.g., embryoninc stem cells into heart) in vitro or in vivo and then be transplanted into another animal and the GPCR reporter may be used for example to track viability, incorporation and trafficking of cells that may migrate from the graft. This is important for tissue engineering for example of a new external ear or liver. I. Stem Cell Production and Storage The invention includes a method of generating stem cells by obtaining cells, stem cells or immune cells from a transgenic animal. For example, bone marrow may be used as a stem cell source and be directly introduced into another animal. Stem cells may be introduced into a subject with other cells in order to improve engraftment. In some embodiments, obtaining stem cells involves fractionating the cells into a fraction enriched with a stem cell and culturing the stem cells in a culture medium containing one or more growth factors. By this process, the stem cells will undergo mitotic expansion. The invention contemplates the establishment and maintenance of cultures of stem cells as well as mixed cultures comprising stem cells, mature cells and mature cell lines. The establishment and maintenance of stem cell cultures involve techniques that are well-known to those of ordinary skill in the art. Once the cells of the invention have been established in culture, they may be maintained or stored in "cell banks" comprising either continuous in vitro cultures of cells requiring regular transfer, or, preferably, cells which have been cryopreserved.

Cryopreservation of cells of the invention may be carried out according to methods known to those of ordinary skill in the art. The cryopreserved cells of the invention constitute a bank of cells, portions of which can be "withdrawn" by thawing and then used to produce new stem cells, etc. as needed. Alternatively, the cells of the invention may be used as ubiquitous donor cells, i.e., to produce new tissue for use in any subject (heterologous). J. Clinical Applications and Pharmaceutical Preparations

1. Clinical Applications "Treatment" and "treating" refer to administration or application of a drug or therapy

(such as protein, nucleic acid, gene therapy, or cell-based therapy) to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition.

The term "therapeutic benefit" used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of his condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease.

A "disease" or "health-related condition" can be any pathological condition of a body part, an organ, or a system resulting from any cause, such as infection, genetic defect, trauma, and/or environmental stress. The cause may or may not be known. Examples of such conditions include cancer and diabetes.

"Prevention" and "preventing" are used according to their ordinary and plain meaning to mean "acting before" or such an act. In the context of a particular disease or health-related condition, those terms refer to administration or application of an agent, drug, or remedy to a subject or performance of a procedure or modality on a subject for the purpose of blocking the onset of a disease or health-related condition. The cells of the invention may be applied to treat subjects requiring the repair or replacement of body tissues resulting from disease or trauma. Treatment may entail the use of the cells of the invention to produce or induce new tissue, and the use of the tissue thus produced, according to any method presently known in the art or to be developed in the future. For example, the cells of the invention may be given systemically, implanted, injected or otherwise administered directly to the site of tissue damage so that they will produce or induce new tissue in vivo.

In addition, the stem cells, the mature cells produced from these stem cells, and the cell lines derived from these stem cells can be used: (1) to screen for the efficacy and/or cytotoxicity of compounds, allergens, growth/regulatory factors, pharmaceutical compounds, etc.; (2) to elucidate the mechanism of certain diseases; (3) to study the mechanism by which drugs operate; (4) to diagnose, monitor and treat cancer in a patient; (5) for gene therapy; and (6) to produce biologically active products, to name but a few uses.

In addition, immune cells can be applied in methods of therapy. These cells may be active against cells expressing a particular antigen. Methods of therapy involving immune cells involve techniques well-known to those of ordinary skill in the art.

Certain embodiments of the present invention involve introducing a pharmaceutically acceptable dose of cells encoding a truncated recombinant GPCR. Pharmaceutical compositions of the present invention comprise a therapeutically or diagnostically effective amount of the cells of the present invention. The phrases "pharmaceutical or pharmacologically acceptable" or "therapeutically effective" or "diagnostically effective" refers to compositions of cells of the present invention that do not produce an unacceptable adverse, allergic or other untoward reaction when administered to a subject, such as, for example, a human or a laboratory animal {e.g., mouse, rat, dog), as appropriate. One of ordinary skill in the art would be familiar with protocols known in the art for the administration of cells (such as stem cells) to a subject for the treatment of a disease. For example, see U.S. Patent 5,139,941, U.S. Patent 5,670,148, U.S. Patent 7,078,032, and U.S. Patent 6,927,060, each of which is hereby specifically incorporated by reference.

2. Pharmaceutical Preparations and Compositions As used herein, "a composition comprising a therapeutically effective amount" or "a composition comprising a diagnostically effective amount" includes any and all solvents, dispersion media, antioxidants, preservatives {e.g., antibacterial agents, antifungal agents), cell culture media, isotonic agents, salts, preservatives, drugs, drug stabilizers, gels, and combinations thereof, as would be known to one of ordinary skill in the art.

The cells of the present invention can be introduced to a subject by any method known to those of ordinary skill in the art. Examples include intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticular Iy, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, directly into a heart chamber, directly injected into the organ or portion of organ or diseased site of interest, or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art.

The actual required amount of a composition of the present invention administered to a subject, such as a patient with a disease, can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

The present invention contemplates methods of preventing, inhibiting, or treating such diseases or conditions in a subject by administration of a cell that has been transfected with an expression construct encoding a truncated recombinant GPCR amino acid sequence operatively linked to a promoter. Aspects of the invention also include the use of the methods and compositions of the invention in combination with other therapies, as discussed in greater detail below.

Diseases to be prevented, treated or diagnosed can be any disease that affect a subject that would be amenable to therapy or prevention through administration of a cell as described herein. For example, the disease may be a disease amenable to stem cell therapy. Examples include cancer, diabetes, cardiovascular disease, neurological disease, neurodegenerative disease, genetic disease, liver disease, infection, trauma, toxicity, or immunological disease. Additional diseases are discussed elsewhere in this specification. For example, the disease may be a hyperproliferative disease. A hyperproliferative disease is a disease associated with the abnormal growth or multiplication of cells. The hyperproliferative disease may be a disease that manifests as lesions in a subject. Exemplary hyperproliferative lesions include pre-malignant lesions, cancer, and tumors. The cancer can be any type of cancer including those derived from mesoderm, endoderm, or ectoderm such as blood, heart, lung, esophagus, muscle, intestine, breast, prostate, stomach, bladder, liver, spleen, pancreas, kidney, neurons, myocytes, leukocytes, immortalized cells, neoplastic cells, tumor cells, cancer cells, duodenum, jejunum, ileum, cecum, colon, rectum, salivary glands, gall bladder, urinary bladder, trachea, larynx, pharynx, aorta, arteries, capillaries, veins, thymus, lymph nodes, bone marrow, pituitary gland, thyroid gland, parathyroid glands, adrenal glands, brain, cerebrum, cerebellum, medulla, pons, spinal cord, nerves, skeletal muscle, smooth muscle, bone, testes, epidiymides, prostate, seminal vesicles, penis, ovaries, uterus, mammary glands, vagina, skin, eyes, or optic nerve.

Other examples of diseases to be treated include return of lost or lack of function such as diabetes where insulin production is inadequate, infectious diseases, genetic diseases, and inflammatory diseases, such as autoimmune diseases. The methods and compositions of the present invention can be applied to deliver an antigen that can be applied in immune therapy or immune prophylaxis of a disease. One of ordinary skill in the art would be familiar with the many disease entities that would be amenable to prevention or treatment using the pharmaceutical compositions and methods set forth herein.

K. Therapy in Combination with Stem Cell Tracking

Particular aspects of the present invention include treating a subject with a disease in combination with the methods for tracking the location of a cell set forth herein. In some aspects, the invention pertains to a method of tracking the location of a cell in a subject and treating a subject with a disease. As set forth above, the disease can be any disease, but in particular embodiments the disease is one for which stem cell therapy is known or suspected to be of benefit.

L. Examples The following examples are included to demonstrate particular embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute particular modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1

Signaling Can Be Uncoupled from Imaging of the Somatostatin Receptor Type 2 Materials and Methods

Cloning of SSTR2A and SSTR2Δ314 inserts. Plasmids for SSTRl, 4, and 5 were obtained from the Donald Guthrie Foundation for Education and Research Inc. (Guthrie cDNA

Resource Center, Sayre, PA) and for SSTR3 from American Type Culture Collection

(ATCC, Rockville, MD). Human SSTR2A and the deletion mutant (SSTR2Δ314) were constructed from a phage (ATCC, Rockville, MD) containing the SSTR2A insert by PCR using the following oligonucleotide primers: SSTR2 FL-F (SEQ ID NO: 23) (TCC CCG CGG CAT GGA CAT GGC GGATGA), SSTR2 FL-R (SEQ ID NO: 24) (AAT CTG CAG

CTG TCA GAT ACT GGT TTG GAG), and SSTR2 T71B-R (SEQ ID NO: 25) (AGA AGG

CAT ATA GGA TAG GGT TGG CAC AGC). The reverse primers contained a Pst I restriction site and a stop codon. The full-length SSTR2A and SSTR2Δ314 inserts were ligated into the Sac II and Pst I inserts in pDisplay vector (Invitrogen, Carlsbad, CA) separately. The inserts were placed downstream of the membrane localization sequence (5 '

IgK leader) and the sequence for hemagglutinin A (HA) epitope tag. The inserts were confirmed by sequencing.

Cell lines. HT 1080 (human fibrosarcoma, ATCC, Rockville, MD) and HEK293

(human embryonic kidney, ATCC, Rockville, MD) cells were maintained in DMEM supplemented with 10% fetal bovine serum and Ix penicillin-streptomycin mixture.

Adherent cultures were incubated at 37 C in a mixture of 5% CO₂ and 95% air. For transfection of HEK293 cells, 2 μg DNA mixed with 3 μl FuGenβ (Invivogen, San Diego, CA) transfection reagent in 50 μl DMEM was added to 1 x 10⁵ cells in 24-well plates per manufacturer's instructions. For HT1080 cells, lipofectin (Invitrogen, Carlsbad, CA) was used per manufacturer's instructions (Kundra et ah, 2002). Cells were selected with 1 mg/ml G418. Enzyme-linked Immunosorbent Assay (ELISA) and Immunofluorescence.

ELISA and immunofluorescence were performed as previously described (Kundra et al, 2002). Prospective colonies expressing fusion protein were screened by ELISA, and expression in selected clones was quantified using a quantitative ELISA. 25mU/ml horseradish peroxidase (HRP) conjugated rat anti-HA antibody (clone 3F10 from Roche, Indianapolis, IN) was used for ELISA. Indirect immunofluorescence employed an anti-HA primary antibody (HA-11, Babco, Richmond, CA).

Reverse transcriptase-polymerase chain reaction (RT-PCR). Total RNA was isolated by extraction with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). Four micrograms of total RNA were reverse transcribed and PCR reactions were carried out with

Superscript One-Step RT-PCR kit (Invitrogen, Carlsbad, CA, USA). PCR was performed with a thermal cycler (Eppendorf, Westbury, NY). The conditions were one cycle each (30 min at 50 C and 2 min at 94 C) for cDNA synthesis and denaturation, 35 cycles (15 s at 94°C, 30 s at 60°C, and 1 min at 72°C) for PCR amplification, and one cycle (5 min at 72°C) for final extension. PCR reactions were carried out in a total volume of 50 μl containing 0.2 μM primers and 2 units of taq in EcoTaq buffer. The primer pairs used SSTR2-FL-F: (SEQ ID NO:26) ATGGACATGGCGGATGAGCCACTCAATGG with SSTR2-FL-R: (SEQ ID NO:27) TCAGATACTGGTTTGGAGGTCTCCATTGAG for the wild-type receptor transcript and SSTR2-F1-F: (SEQ ID NO:28) ATGGACATGGCGGATGAGCCACTCAATGG with SSTR2-Δ314-R: (SEQ ID NO:29) AGAAGGCATATAGGATAGGGTTGGACAGC for the portion of the transcript coding up to amino acid 314. Primers described previously for SSTR 1, 3, 4, and 5 (Panetta et al, 1995), as well as SSTR2-FL-F and SSTR2-Δ314-R were used for RT-PCR of untransfected HEK293 and HT1080 cells. Ten microliters of each PCR reaction were subjected to electrophoresis on a 1% agarose gel that was stained with ethidium bromide and visualized under ultraviolet light.

Binding assay. Cells were harvested in binding buffer (50 mM Tris-HCl, pH 7.8, containing 1 mM EGTA, 5 mM MgCl₂, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 200 μg/ml bacitracin, and 0.5 μg/ml aprotinin) and were centrifuged at 12,000 rpm for 10 min at 4 C. The pellet was homogenized with a polytron at 50 Hz for 20 s in binding buffer. Five micrograms per well of the membrane preparation was used for the radioligand binding studies (Birzin et al, 2002). Each well of the GF/B multi-screen plates (Millipore, Bedford, MA) was pretreated with 100 μl of 0.1% polyethyleneimine per well for 2 h. The solution was removed using a multiscreen vacuum manifold (Millipore, Bedford, MA) and then the filter plates were washed one time with 200 μl of 50 mM Tris-HCI (pH 7.8). Assay reagents were added to the washed plates in the following order: 160 μl membrane and 40 μl (200 nM-0.7 nM final concentration) ¹¹¹In-octreotide (Mallinckrodt, St. Louis, MO), or 160μl membrane, 20 μl unlabeled ligand (1 μM final concentration of unlabeled somatostatin), 20μl ^ulIn-octreotide (200 nM-0.7 nM final concentration). The assay was run in triplicate. Plates were incubated at room temperature for 40 min. The solution was filtered from the plate and washed two times with 350 μl of unlabeled 50 mM Tris-HCI (pH 7.8). The filters were punched out from the plates. The radioactivity of each filter was quantified with a γ-counter. Scatchard analysis was performed.

For radioligand uptake, 30,000 cells/well were exposed to 10^"7 M ^{l u}In-octreotide with or without 10^"6 M somatostatin for varying amounts of time at 37°C. The cells were then washed three times with PBS and the radioactivity associated with the cells was measured by a gamma counter. The experiment was performed in triplicate.

Determination of cAMP or cGMP levels. Intracellular levels of cAMP or cGMP in response to somatostatin were measured in triplicate by modified ELISA methods. For measuring cAMP, the cells were incubated at 37 C for 30 min in 1 ml PBS with or without forskolin (10 ⁷ M), or somatostatin (10^~n - 10^"7M) plus forskolin (10⁷ M). For cGMP detection, the cells were incubated with or without somatostatin (10 ¹¹ - 10^"7M). The levels of cAMP or cGMP in the cell lysates were detected using commercially available ELISA kits (Sigma-Aldrich, St. Louis, MO). The concentrations of cAMP or cGMP were calculated from standard curves.

Growth inhibition assay. Cells (3000 cells/well) were seeded in 96-well plates in 5% serum containing medium and treated in the same medium with or without Sandostatin

(10 ¹¹ - 10^"7M) in triplicate for another 2 days. MTT (20 μl, Promega, Madison, WI) was added to each well. After an one-hour incubation at 37 C, the color reaction was quantified using an automatic plate reader (VERSAmax, Sunnyvale, CA) at 490 nm.

Imaging. The animal protocol was approved by the institutional animal care and use committee. Eight- to ten-week old female nude mice bred within the institution were utilized.

To produce tumors, 5 x 10⁶ cells were injected subcutaneous Iy. Each mouse received three inoculations: left thigh, HT1080 cells transfected with vector; left shoulder, HT1080 cells expressing wild-type fusion protein; and right shoulder, HT 1080 cells expressing Δ314 fusion protein. After approximately one and one-half weeks, imaging and bio distribution analysis was performed as described (Yang et al, 2005). The mice were injected intravenously via the tail vein with 300 μCi of ^luIn-octreotide (Mallinckrodt, St. Louis, Mo). The same day, the mice were imaged with a 4.7 T small animal MR (Bruker, Billerica, MA) using a T2- weighted fast spin echo sequence. Twenty-four hours after radiopharmaceutical injection, the animals were imaged for 10 min using a γ-camera fitted with a medium-energy collimator (mCAM, Siemens Medical Solutions, Hoffman Estates, IL). Acquisition was with a 512 x 512 matrix that was compressed to a 256 x 256 matrix for viewing and measurement. A combination of the in vivo MR and γ-camera imaging was used to determine the biodistribution of the radiopharmaceutical in tumors.

Statistical Methods. For ELISA, receptor binding, radioligand uptake, cAMP, cGMP, and MTT assays, as well as biodistribution analysis, two sided Student's t-tests were performed using Excel (2000, Microsoft, Redmond, WA). For all experiments, a P value of less than 0.05 was considered statistically significant.

Results

Creation of cell lines expressing similar levels of wild type or mutant receptors.

Human embryonic kidney (HEK 293) and human fibrosarcoma (HT 1080) cells do not endogenously express somatostatin receptors. As shown in FIG. IA, no SSTR subtype expression was found using RT-PCR of RNA extracts from either cell line. RT-PCR of the same RNA extracts did result in a product for the ubiquitiously expressed β-actin, confirming the quality of the RNA. Functionality of the SSTR subtype primers was confirmed by PCR of SSTR subtype cDNA.

To inhibit signaling by the SSTR2A, the intracytoplasmic portion of the carboxy- terminus was deleted beyond amino acid 314 (Δ314). Gene chimeras consisting of an Igκ membrane localization sequence, HA epitope tag and full length (wt) or Δ314 SSTR2 were constructed. These were used to clone stable transfectants in both human embryonic kidney

(HEK 293) and human fibrosarcoma (HT 1080) cells. Reverse transcriptase polymerase chain reaction (RT-PCR) of cellular RNA extracts confirmed expression of appropriate transcripts. Primers for either wild type receptor or the Δ314 mutation resulted in appropriately sized products when RNA from either HEK293 or HT 1080 cells transfected with wild type receptor was used. In comparison, only primers for Δ314 resulted in appropriately sized products when RNA from HEK293 or HT 1080 cells transfected with the Δ314 receptor was used; whereas, no product was seen when primers for the full length receptor were used. Thus, HEK293 or HT1080 clones transfected with wild-type or Δ314 SSTR2 gene chimeras express the predicted RNA transcripts. Immunofluorescence demonstrated expression of wild-type or Δ314 SSTR2 fusion protein on the cell membranes of transfected HT 1080 or HEK293 clones, and background signal on vector transfected cells.

To assess expression at the protein level, a quantitative enzyme linked immunosorbent assay (ELISA) was utilized. The common epitope tag was targeted instead of sites in the receptors in order to avoid confounding results that may arise from potential changes in ligand or antibody affinity due to receptor mutation. Using an antibody to the HA tag, similar levels of fusion protein expression were seen in cells transfected with wild type receptor or the Δ314 deletion mutant for pairs of either HTl 080 (as shown in FIG. IB) or HEK293 (as shown in FIG. 1C) cells. Background signal was seen in cells expressing vector. The data demonstrate that the HEK293 or HT 1080 clonal pairs express similar levels of wild type or Δ314 fusion protein.

Receptor-ligand binding. The hypothesis that the cytoplasmic carboxy-terminus beyond amino acid 314 of human SSTR2 is not essential for ligand binding was next tested. Studies were conducted to examine whether the cytoplasmic carboxy-terminus of human SSTR2 is essential for detecting the receptor in vivo. The imaging agent ^{l u}In-octreotide was used in binding experiments to evaluate a ligand relevant to in vivo imaging. Scatchard analysis demonstrated similar degrees of binding to membranes from clonal pairs expressing either full length or Δ314 SSTR2 receptors, as shown in FIG. 2A, FIG. 2B, FIG. 2C, and FIG. 2D. Similar results, with IQ in the nanomolar range, were seen whether HEK293 or HT 1080 cell trans fectants were evaluated; thus, binding to the imaging agent ^ulIn-octreotide is conserved in the HA-SSTR2Δ314 receptor.

No significant difference in uptake of ^luIn-octreotide was appreciated between HT1080 or HEK293 clonal pairs expressing wild-type or Δ314 SSTR2 receptors, as shown in FIG. 2E. Unlabeled somatostatin inhibited uptake as expected. Biochemical activity, signaling. To assess receptor function, two separate signaling pathways were evaluated. Studies were first conducted to assess the ability of the human Δ314 SSTR2 mutant to inhibit cAMP production. No difference in baseline cAMP was noted in the cell line pairs expressing wild type or Δ314 fusion protein (p<.05), see FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D. To activate adenylate cyclase, forskolin was utilized. Addition of forskolin to HEK293 or HT 1080 cells resulted in cAMP production whether the cells expressed wild type or Δ314 fusion protein. Addition of somatostatin inhibited forskolin- induced production of cAMP in either cell line expressing full length receptors. In comparison, somatostatin did not inhibit forskolin-induced production of cAMP in HEK293 or HT 1080 cells expressing the Δ314 mutant. Findings in two different human cell types imply that the human HA-SSTR2Δ314 mutant is deficient in regulating the cAMP signaling pathway. Studies were then conducted to determine whether deleting amino acids distal to amino acid 314 affects the capacity of the SSTR2 to activate another signaling pathway, namely, incitement of cGMP production, as shown in FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D. No difference in baseline cGMP was noted in the cell line pairs expressing wild type or Δ314 fusion protein (p < 0.05). Addition of somatostatin to HEK293 or HT1080 cells expressing wild type receptors resulted in increased cGMP production. In comparison, somatostatin did not cause cGMP production in HEK293 or HT 1080 cells expressing the Δ314 mutant. The findings suggest that when expressed in HEK293 cells or HT 1080 cells, the Δ314 mutant is deficient in regulating the cGMP signaling pathway. Thus, biochemical signaling pathways regulating two different downstream mediators, cAMP or cGMP, are deficient in the Δ314 SSTR2 mutant.

Cellular activity, mitosis. Function of the Δ314 mutant was further assessed at the cellular level. Whether deleting amino acids distal to 314 affects the capacity of the SSTR2A to inhibit proliferation was tested in HEK293 and HT 1080 cells, as shown in FIG. 5 A, 5B, 5C, and 5D. Somatostatin is not stable in culture media long term, therefore, a stabilized analogue, Sandostatin, was used to maintain receptor activation. Serum stimulation promoted growth in HEK293 cells or HT 1080 cell clones. As predicted, Sandostatin inhibited serum- induced proliferation in HEK293 cells or HT 1080 cells expressing full length receptors. In contrast, HEK293 or HT 1080 cells expressing the Δ314 mutants were muted in inhibiting serum-induced proliferation. Thus, the Δ314 SSTR2 mutant is deficient in inhibiting cellular proliferation.

In vivo imaging. The SSTR2Δ314 mutant was utilized to evaluate whether in vivo imaging of SSTR2 can be uncoupled from cAMP and cGMP mediated signal transduction as well as growth inhibition. Nude mice were injected subcutaneously with transfected cells. HT 1080 cells were used because they reliably produce tumors in nude mice. Once tumors formed, the animals were injected with 300 μCi of the somatostatin analogue ¹¹¹In octreotide and underwent planar gamma camera imaging the next day. Upon imaging, both tumors derived from cells expressing Δ314 and tumors derived from cells expressing wild-type receptor were clearly visible. In comparison, tumors derived from cells transfected with vector were not seen. For ^ulIn-octreotide, the normal route of excretion is primarily renal with a secondary route via the hepatobiliary system. As expected, physiologic excretion of the radiopharmaceutical resulted in prominence of the kidneys. The data imply that the SSTR2Δ314 mutant is competent for imaging in vivo.

Radiopharmaceutical uptake by tumors was quantified by in vivo biodistribution analysis (O'Carroll et ah, 1993). To control for tumor weight, prior to gamma camera imaging, the animals underwent T2 -weighted fast spin echo (FSE) magnetic resonance (MR) imaging to obtain the weight of each tumor. Tumors expressing either wild-type receptor or the Δ314 mutant had greater uptake of ^{l u}In-octreotide than tumors derived from cells transfected with vector (P < 0.05, n = 6; FIG. 6). No significant difference in uptake was seen in tumors expressing either wild-type receptor or the Δ314 mutant. The biodistribution analysis confirmed the imaging findings that the SSTR2Δ314 receptor is amenable to imaging in vivo. Planar γ-camera images of a nude mouse demonstrated that tumors derived from HT1080 cells expressing HA-wt SSTR2 or expressing HA-SSTR2Δ314 were visible. In contrast, the tumor derived from HT 1080 cells transfected with vector was not visible.

EXAMPLE 2

Studies Using Human Bone Marrow Mesenchymal Cells Materials and Methods Cell lines. HS-5 (human bone marrow messencymal cells, ATCC, Rockville, MD) were maintained in α-MEM supplemented with 10% fetal bovine serum and Ix penicillin- streptomycin mixture. Adherent cultures were incubated at 37 C in a mixture of 5% CO₂ and 95% air. Gene transfer was performed using nucleofection.

Binding assay. The binding assay was as described in Example 1 above except a single dose (10^"7M) of ^{l u}In-octreotide was used. Competition was performed using 10 ⁶ M somatostatin. Growth Inhbition Assay. The growth inhibition assay was as described in Example 1 except a single dose (10^"7M) of ^{l u}In-octreotide was used.

Osteogenic induction: Cells were induced using osteogenic induction medium using the manufacturers instructions (Cambrex, East Rutherford, NJ) and calcium production was evaluated using the StanbioTotal Calcium LiquiColor using the manufacturers instructions (Stanbio Laboratory, Boerne, TX).

Adipogenic induction: Cells were induced using the adipogenic induction medium using the manufacturers instructions (Cambrex, East Rutherford, NJ). After induction, the cells were rinsed in phosphate buffered saline (PBS), fixed in 10% buffered formalin and stained with Oil Red O to visualize lipid vacuoles.

Imaging. 10⁶ HS-5 cells stably transfected with HA-SSTR2Δ314 HA-wt SSTR2, or vector were exposed to 10^"7 M ¹¹¹In octreotide for two hours, washed and then injected into C57/B16 mice via tail vein. No cell control incubated with 10^"7 M ¹¹¹In octreotide for two hours was washed and then injected into another C57/B16 mouse via tail vein. Imaging was performed as described in Example 1 above for planar gamma camera acquisition except counts were collected from 10 to 60 minutes post injection.

Results

Results of a quantitative ELISA using an antibody to the HA-domain showed that stably transfected HS-5 cells express similar amounts of HA-SSTR2Δ314 (A314) or HA- wtSSTR2 (wt) (FIG. 7). Further, it was found that stably transfected HS-5 cells expressing A314 or HA-wt SSTR2 (wt) bind similar amounts of ¹¹¹In octreotide (FIG. 8).

It was found that HA-SSTR2Δ314 is signaling deficient for the cAMP pathway in human bone marrow mesenchymal cells, HS-5 (FIG. 9). Upon ligand (100 nM somatostatin- 14) binding, HA-wt SSTR2 decreased forskolin-induced cAMP production (wt) whereas HA- SSTR2Δ314 (A314) did not when expressed in HS-5 cells. HA-SSTR2Δ314 was found to be signaling deficient for the cGMP pathway in human bone marrow mesenchymal cells, HS-5 (FIG. 10). Upon ligand (100 nM somatostatin- 14) binding, HA-wt SSTR2 (wt) incited cGMP production, whereas HA-SSTR2Δ314 (Δ314) does not when expressed in HS-5 cells.

Further, HA-SSTR2Δ314 (A314) was found to be deficient in inhibiting cell growth in human bone marrow mesenchymal cells, HS-5 (FIG. 11). Upon ligand (100 nM Sandostatin) binding, HA-wt SSTR2 (wt) decreased proliferation induced by serum, whereas HA-SSTR2Δ314 (A314) did not when expressed in HS-5 cells (P < 0.05). Human bone marrow mesenchymal cells, HS-5, expressing HA-SSTR2Δ314 (Δ314) differentiate into osteoclast lineage cells that produce calcium phosphate (FIG. 12). Cells transfected with vector, HA-SSTR2Δ314 (Δ314), or HA-wt SSTR2 (wt) produced calcium phosphate upon exposure to osteogenic induction medium. In osteoclaset differentiation medium, calcium phosphate production was equivalent in cells transfected with vector or HA-SSTR2Δ314, but decreased in cells transfected with HA-wt SSTR2 (FIG. 12).

It was further found that human bone marrow mesenchymal cells, HS-5, expressing HA-SSTR2Δ314 differentiate into adipocyte lineage. Cells transfected with vector, HA-wt SSTR2, or HA-SSTR2Δ314, demonstrated increased staining for fat using Oil Red O with exposure to adipogenic induction medium compared to without exposure.

Using planar gamma camera imaging, in vivo imaging of cell trafficking of human bone marrow mesenchymal cells, HS-5, expressing HA-SSTR2Δ314, was performed (FIG. 14). HS-5 cells stably transfected with HA-SSTR2Δ314, HA-wt SSTR2, or vector were exposed to 10^~7 M ^luIn-octreotide for two hours, washed and then injected into C57/B16 mice via tail vein. A control that did not include cells was incubated with 10^~7 M ^luIn-octreotide for two hours, washed and then injected into C57/B16 mice via tail vein. Increased signal was found to overlie the lungs in mice injected with HS-5 cells expressing HA-SSTR2Δ314 or HA-wt SSTR2HS-5. The planar gamma camera image was collected from 10 to 60 minutes post injection. FIGS. 15-20 demonstrate that cells expressing the SSTR2Δ314 without a tag behave similarly to those with the tag. A tag can be important, for example, for differentiating endogenous from exogenous SSTR2 expression and for reducing costs of in vitro and in vivo assessment, but may not be desirable under some circumstances. In some embodiments, a SSTR2Δ314 reflecting only human-derived sequences may be advantageous, for example, to reduce the possibility of an immune reaction to a tag or to decrease the already small size of the insert (of the reporter gene) in a construct. EXAMPLE 3

Studies Using Isolated Human Peripheral White Blood Cells Materials and Methods

Human peripheral white blood cells. Human peripheral white blood cells were isolated via a Ficoll gradient. Each 10 ml of blood collected in a heparinized tube was mixed with 25 ml of phosphate buffered saline without calcium or magnesium and layered upon 15 ml of Histopaque-1077 (Sigma- Aldrich, St. Louis, MO) in a 50 ml conical tube and centrifuged at 4°C at 400 x g for 20 minutes. The buffy coat was resuspended in PBS and centrifuged at 4°C at 400 x g for 20 minutes. The cells were then washed again with PBS and transferred to a flask containing α-MEM with 20% fetal calf serum. The cells were grown in a humidified 5% CO₂ incubator at 37°C. Some cells were transferred to a slide and stained with Hematoxylin. The white blood cells were infected with 10¹¹ adenovirus particles containing an insert for HA-wt SSTR2, HA-SSTR2Δ314, or control. Expression was confirmed by ELISA. Imaging. 10⁶ white blood cells infected with HA-SSTR2Δ314 HA-wt SSTR2, or vector were exposed to 10^"7 M ¹¹¹In octreotide for two hours, washed and then injected into nude mice via tail vein. No cell control incubated with 10^"7 M ¹¹¹In octreotide for two hours was washed and then injected into another nude mouse via tail vein. Imaging was performed as described in Example 1 above for planar gamma camera acquisition except counts were collected from 10 to 60 minutes post injection.

Results

Hematoxylin staining demonstrated nuclei and confirmed isolation of white blood cells (FIG. 21).

Expression of HA-SSTR2Δ314 by isolated human peripheral white blood cells. It was found that isolated human peripheral blood white blood cells infected with adenovirus containing an insert for HA-SSTR2Δ314 (Δ314) or HA-wt SSTR2 (wt) express the fusion proteins. Results of quantitative ELISA using an antibody to the HA-domain are shown in FIG. 22. Expression of HA-wt SSTR2 (wt) was greater than that of HA-SSTR2Δ314 (Δ314) in the sets of cells used for the cell trafficking experiments discussed below. In vivo imaging of cell trafficking of isolated human peripheral white blood cells expressing HA - SSTR2Δ314. White blood cells were infected with adenovirus containing an insert for HA-wt SSTR2, HA-SSTR2Δ314, or control were exposed to 10^~7 M ¹¹¹In- octreotide for two hours, washed and then injected into nude mice via tail vein. A no cell control was incubated with 10^~7 M ^ulIn-octreotide for two hours, washed and then injected into nude mice via tail vein. Increased signal was found to overlie the lungs in mice injected with white blood cells infected with adenovirus containing an insert for HA-SSTR2Δ314 or HA-wt SSTR2 (FIG. 23). Expression of HA-wt SSTR2 was greater than that of HA-SSTR2Δ314 in these sets of cells used for the cell trafficking experiment (see FIG. 22) and this is reflected in the imaging. The planar gamma camera image was collected from 10 to 60 minutes post injection. EXAMPLE 4

Transgenic Mice Expressing HA-SSTR2A314 Materials and Methods

Transgenic mice production. A construct containing a ubiquitin promoter-intron-HA- SSTR2Δ314-bovine growth hormone polyA was introduced into B6D2F1 mouse oocytes using pronuclear injection. Mice were bred in order to obtain homozygotes.

Transgenic mice characterization. RT-PCR was performed of different mouse organs as described in Example 1 above.

Western blotting was performed using an antibody to the HA tag. Mouse organs were homogenized with a glass mortar 20 times in Tris/SDS lysis solution (1% SDS, 10 mM Tris pH7.4, 1 mM Sodium ortho-vandate) heated to 100⁰C before use. The tissue lysate was boiled for 3 minutes at 100⁰C and then cooled on ice. After centrifugation at 12,000 rpm in an Eppendorf Micro fuge for 10 minutes, the supernatant was collected for determination of protein concentration using the Bradford method (Bio-Rad Laboratories, Hercules, CA). 20 micrograms of protein boiled in loading buffer was loaded per lane on a 9% SDS gel. Western blotting was performed using an overnight exposure at 4°C to a mouse anti HA-I l antibody (1 :2000 dilution, Covance, Berkley, CA). After washing, an HRP-conjugated secondary goat-antimouse IgG antibody (1 :4000 dilution, Biorad Laboratories, Hercules, CA) was used for detection.

Results Exemplary constructs incorporating HA-SSTR2Δ314 are shown in FIG. 24. FIG.

24A shows examples of constructs using constitutive promoters (viral-CMV, and human - ubiquitin). FIG. 24B shows a construct incorporating a functional and tissue-selective promoter as a marker for hepatocytes, since albumin expression is essentially restricted to hepatocytes. FIG. 24C shows an example of an amplified functional and tissue-selective promoter (miniCMV) for amplifying expression from the albumin promoter. Introns need not be used, but can be helpful to increase expression in transgenic animals, and their positions in the construct may be varied.

Transgenic mice were created using a construct with an ubiquitin promoter for driving expression of HA-SSTR2Δ314. Transgenic mice were found to express HA-SSTR2Δ314 mRNA in multiple organs (FIG. 21). FIG. 25 demonstrates results of a reverse transcriptase- polymerase chain reaction of RNA derived from transgenic mice or non-transgenic parental strain mice. Primers for HA-SSTR2Δ314 demonstrated expression of HA-SSTR2Δ314 in only transgenic mice.

It was also found that the transgenic mice express HA-SSTR2Δ314 protein in multiple organs, including bone marrow. Results of Western blotting of protein derived from transgenic mice or non-transgenic parental strain mouse demonstrated expression of HA- SSTR2Δ314 in only transgenic mice (FIG. 26).

FIG. 27 demonstrates specific expression of HA-SSTR2Δ314 protein in the liver of the transgenic mouse. No expression is seen in the wild type mouse. FIG. 27 shows that tissue-specific expression can be obtained with the signaling deficient reporter, HA- SSTR2Δ314. This is important, for example, for evaluating context specific expression and differentiation such as of stem cells, and for creating transgenic mice that express the signaling deficient reporter in specific tissues.

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Claims

1. A method for tracking the location of a cell in a subject, comprising:

a) obtaining a cell;

b) transferring into the cell an expression construct comprising a first coding region encoding a first reporter comprising a truncated recombinant seven transmembrane G- protein associated receptor (GPCR) amino acid sequence operatively linked to a first promoter sequence;

c) contacting the cell with a detectable moiety that binds to the first reporter;

d) introducing the cell to the subject; and

e) imaging the detectable moiety using an imaging technique.

2. The method of claim 1, further defined as a method for tracking the location of progeny of the cell in a subject, further comprising contacting progeny of the cell with a detectable moiety that binds to the first reporter that is encoded in the progeny.

3. The method of claim 1, wherein the cell is a stem cell or an immune cell.

4. The method of claim 3, wherein the cell is a stem cell further defined as a stem cell selected from the group consisting of an embryonic stem cell, a somatic stem cell, a germ stem cell, an epidermal stem cell, and a tissue-specific stem cell.

5. The method of claim 4, wherein the stem cell is a tissue-specific stem cell.

6. The method of claim 5, wherein the tissue-specific stem cell is selected from the group consisting of a cancer stem cell, an adult neural stem cell, a human neuron, a human oligodendrocyte, a human astrocyte, a human keratinocyte stem cell, a human keratinocyte transient amplifying cell, a human melanocyte stem cell, a human melanocyte, a human foreskin fibroblast, a human duct cell, a human pancreatic islet, a human pancreatic β-cell, a human adult renal stem cell, a human embryonic renal epithelial stem cell, a human kidney epithelial cell, a human hepatic oval cell, a human hepatocytes, a human bile duct epithelial cell, a human embryonic endodermal stem cell, a human adult hepatocyte stem cell (controversial as to existence), a human mammary epithelial stem cell, bone marrow-derived stem cell, a human lung fibroblasts, a human bronchial epithelial cell, a human alveolar type II pneumocyte, a human skeletal muscle stem cell (satellite cell), a human cardiomyocyte, bone marrow mesenchymal stem cell, simple squamous epithelial cell, descending aortic endothelial cell, aortic arch endothelial cell, aortic smooth muscle cell, limbal stem cell, corneal epithelial cell, CD34+ hematopoietic stem cell, mesenchymal stem cell, osteoblast (precursor is mesenchymal stem cell), peripheral blood mononuclear progenitor cell, osteoclast, stromal cell, a human splenic precursor stem cell, a human splenocyte, a human CD4+ T-cell, a human CD8+ T-cell, a human NK cell, a human monocyte, a human macrophage, a human dendritic cell, a human B-cell, goblet cell, pseudostriated ciliated columnar cell pseudostriated epithelium stratified epithelial cell, ciliated columnar cell, goblet cell, basal cell, cricopharyngeus muscle cell, oesophageal stem cell, oesophageal transit amplifying cell, female primary follicle, and male spermatogonium.

7. The method of claim 3, wherein the stem cell is an autologous stem cell.

8. The method of claim 3, wherein the stem cell is an allogeneic stem cell.

9. The method of claim 3, wherein the stem cell is a xenogeneic stem cell.

10. The method of claim 1, wherein the recombinant GPCR has a C-terminal deletion.

11. The method of claim 1, wherein the recombinant GPCR has altered signaling including is signaling defective, has altered internalization, or a combination thereof.

12. The method of claim 1, wherein the GPCR is an acetylcholine receptor: Ml, M2, M3, M4, or M5; adenosine receptor: Al; A2A; A2B; or A3; adrenoceptors: alphalA, alphalB, alphalD, alpha2A, alpha2B, alpha2C betal, beta2, or beta3; angiotensin receptors: ATI, or AT2; bombesin receptors: BBl, BB2, or BB3; bradykinin receptors: Bl, B2, calcitonin, Ainilin, CGRP, or adrenomedullin receptors; cannabinoid receptors: CBl, or CB2; chemokine receptors: CCRl, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCRlO, CXCRl, CXCR2, CXCR3, CXCR4, CXCR5, CX3CR1, or XCRl; chemotactic receptors : C3a, C5a, or fJVILP; cholecystokinin and gastrin receptors: CCKl, or CCK2; corticotropin-releasing factor receptors: CRFl, or CRF2; dopamine receptors: Dl, D2, D3, D4, or D5; endothelin receptors: ET(A) or ET(B); galanin receptors: GALl, GAL2, or GAL3; glutamate receptors: mgll, mgl2, mgl3, mgl4, mgl5, mgl6, mgl7, or mgl8; glycoprotein hormone receptors: FSH, LSH, or TSH; histamine receptors: Hl, H2, H3, or H4; 5-HT receptors: 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1B, 5-HT1F, 5HT2A, 5-HT2F, 5-HT2C, 5-HT3, 5-HT4, 5-HT5A, 5-HT5B, 5-HT6, or 5-HT7; leukotriene receptors: BLT, CysLTl, or CysLT2; lysophospholipid receptors: edgl, edg2, edg3, or edg4; melanocorlin receptors: MCl; MC2; MC3; MC4, or MC5; melatonin receptors: MTl, MT2, or MT3; neuropeptide Y receptors: Yl, Y2, Y4, Y5, or Y6; neurotension receptors: NTSl, or NTS2; opioids: DOP, KOP, MOP, or NOP; P2Y receptors: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, or P2Y12); peroxisome proliferators: PPAR-alpha, PPAR-beta, or PPAR-gamma; prostanoid receptors: DP, FP, IP, TP, EPl, EP2, EP3, or EP4; protease-activated receptors: PARl, PAR2, PAR3, or PAR4; Somatostatin receptors: SSTRl, SSTR2, SSTR2A, SSTR3, SSTR4, or SSTR5; tachykinin receptors: NKl, NK2, or NK3; thyrotropin-releasing hormone receptors: TRHl, or TRH2; urotensin-II receptor; vasoactivate intestinal peptide or pituitary adenylate cyclase activating peptide receptors: VPACl, VPAC2, or PACl; or vasopressin or oxytocin receptors: Via, VIb, V2, or OT.

13. The method of claim 12, wherein the GPCR is a somatostatin receptor.

14. The method of claim 13, wherein the somatostatin receptor is a somatostatin receptor type 2A (SSTR2A) or a somatostatin receptor type 2Δ314.

15. The method of claim 14, wherein the SSTR2A or somatostatin receptor type 2Δ314 has altered signaling including is signaling defective, has altered internalization, or a combination thereof.

16. The method of claim 1, wherein the promoter sequence is a constitutive promoter sequence, a tissue-selective promoter sequence, or a function-specific promoter sequence.

17. The method of claim 16, wherein the promoter sequence is a function-specific promoter sequence.

18. The method of claim 17, wherein the function-specific promoter sequence is an insulin promoter sequence, T cell receptor promoter sequence, immunoglobulin promoter sequence, a vascular endothelial growth factor promoter sequences, a dystrophin promoter sequence, a pBROAD promoter sequence, a c-fos promoter sequence, a c-HA-ras promoter sequence, an intercellular adhesion molecule 2 promoter sequence, or a platelet-derived growth factor (PDGF) promoter sequence.

19. The method of claim 16, wherein the promoter sequence is a constitutive promoter sequence.

20. The method of claim 19, wherein the constitutive promoter sequence is a beta-actin promoter sequence, an elastase I promoter sequence, a metallothionein (MTII) promoter sequence, a 5 S ribosomal promoter sequence, an Elastase promoter sequence, an Elastase I promoter sequence, a polyoma promoter sequence, a Cytomegalovirus promoter sequence, a retrovirus promoter sequence, a papilloma virus promoter sequence, a fϊbronectin promoter sequence, a ubiquitin promoter, an actin promoter, an elongation factor 1 alpha, an early growth factor response 1, an eukaryotic initiation factor 4Al, a ferritin heavy chain, a ferritin light chain, a glyceraldehyde 3-phosphate dehydrogenase, a glucose-regulated protein 78, a glucose-regulated protein 94, a heat shock protein 70, a heat shock protein 90, a beta-kinesin, a phosphoglycerate kinase, an ubiquitin B, a beta-actin, RNA virus promoter, DNA virus promoter, adenoviral promoter sequence, a baculoviral promoter sequence, a CMV promoter sequence, a parvovirus promoter sequence, a herpesvirus promoter sequence, a poxvirus promoter sequence, an adeno-associated virus promoter sequence, a semiliki forest virus promoter sequence, an SV40 promoter sequence, a vaccinia virus promoter sequence, a lentivirus promoter, a retrovirus promoter sequence, or a minimal viral promoter sequence.

21. The method of claim 20, wherein the minimal viral promoter sequence is a RNA virus promoter, DNA virus promoter, adenoviral promoter sequence, a baculoviral promoter sequence, a CMV promoter sequence, a parvovirus promoter sequence, a herpesvirus promoter sequence, a poxvirus promoter sequence, an adeno-associated virus promoter sequence, a semiliki forest virus promoter sequence, an SV40 promoter sequence, a vaccinia virus promoter sequence, a lentivirus promoter, or a retrovirus promoter sequence.

22. The method of claim 16, wherein the promoter sequence is a tissue selective promoter sequence.

23. The method of claim 22, wherein the tissue-selective promoter sequence is active in normal and/or diseased heart, lung, esophagus, muscle, intestine, breast, prostate, stomach, bladder, liver, spleen, pancreas, kidney, neurons, myocytes, leukocytes, immortalized cells, neoplastic cells, tumor cells, cancer cells, duodenum, jejunum, ileum, cecum, colon, rectum, salivary glands, gall bladder, urinary bladder, trachea, larynx, pharynx, aorta, arteries, capillaries, veins, thymus, lymph nodes, bone marrow, pituitary gland, thyroid gland, parathyroid glands, adrenal glands, brain, cerebrum, cerebellum, medulla, pons, spinal cord, nerves, skeletal muscle, smooth muscle, bone, testes, epidiymides, prostate, seminal vesicles, penis, ovaries, uterus, mammary glands, vagina, skin, eyes, or optic nerve.

24. The method of claim 22, wherein the promoter sequence is an hTR promoter sequence, hTERT promoter sequence, CEA promoter sequence, a PSA promoter sequence, a probasin promoter sequence, a ARR2PB promoter sequence, AFP promoter sequence, a MUC-I promoter sequence, a MUC-4 promoter sequence, a mucin- like glycoprotein promoter sequence, a C-erbB2/neu oncogene promoter sequence, a cyclo-oxygenase promoter sequence, a E2F transcription factor 1 promoter sequence, a tyrosinase related protein promoter sequence, a tyrosinase promoter sequence, a survivin promoter sequence, a Tcfl -alpha promoter sequence, a Ras promoter sequence, a Raf promoter sequence, a cyclin E promoter sequence, a Cdc25A promoter sequence, a HK II promoter sequence, a KRT 19 promoter sequence, a TFFl promoter sequence, a SELlL promoter sequence, a CEL promoter sequence, an immunoglobulin heavy chain promoter sequence, an immunoglobulin light chain promoter sequence, a T-cell receptor promoter sequence, an HLA DQ a promoter sequence, an HLA DQ beta promoter sequence, a b eta-inter feron promoter sequence, an interleukin-2 promoter sequence, an interleukin-2 receptor promoter sequence, an MHC Class II 5 promoter sequence, an MHC Class II HLA-Dra promoter sequence, a muscle creatine kinase (MCK) promoter sequence, a prealbumin (transthyretin) promoter sequence, an albumin promoter sequence, an alpha-fetoprotein promoter sequence, a gamma-globin promoter sequence, or a beta-globin promoter sequence, an insulin promoter sequence, a neural cell adhesion molecule (NCAM) promoter sequence, an alpha- 1 -antitrypsin promoter sequence, a growth hormone promoter sequence, a human serum amyoid A (SAA) promoter sequence, a troponin I (TN I) promoter sequence, a Duchenne Muscular Dystrophy promoter sequence, an SV40 promoter sequence, a, a Hepatitis B virus promoter sequence, Gibbon Ape Leukemia Virus promoter sequence, a a somatostatin receptor promoter sequence, a human CD4 promoter sequence, a human alpha-lactalbumin promoter sequence, a human Y promoter sequence, alpha fetoprotein, monocyt receptor for bacterial LPS, leukocyte common antigen, Desmin, VEGF receptors, glial fibrillary acidic protein, interferon beta, myoglobin, osteocalcin 2, prostate specific antigen, prostate specific membrane antigen, surfactant protein B, Synapsin, tyrosinase related protein, tyrosinase, a functional hybrid, functional portion, or a combination of any of the aforementioned promoter sequences.

25. The method of claim 1, wherein the nucleic acid further comprises a second coding region.

26. The method of claim 25, wherein a second promoter sequence is operatively linked to the second coding region.

27. The method of claim 25, wherein the first coding region and the second coding region are linked by an IRES or a bidirectional promoter sequence.

28. The method of claim 26, wherein the first promoter sequence and the second promoter sequence are individually selected from the group consisting of a constitutive promoter sequence, a tissue-specific promoter sequence, a lineage-specific promoter, and a function- specific promoter sequence.

29. The method of claim 25, wherein the second coding region comprises a reporter sequence, a therapeutic gene, or signalling sequence, or a trafficking sequence.

30. The method of claim 25, wherein the nucleic acid further comprises a third coding region.

31. The method of claim 30, wherein the third coding region is operatively linked to a third promoter sequence.

32. The method of claim 30, wherein the first coding region, the second coding region, and the third coding region are independent or operably linked by one or more IRES or bidirectional promoter sequences.

33. The method of claim 31, wherein the first promoter sequence, the second promoter sequence, and the third promoter sequence are individually selected from the group consisting of a constitutive promoter sequence, a tissue-specific promoter sequence, a lineage-specific promoter, and a function-specific promoter sequence.

34. The method of claim 30, wherein the first coding region, the second coding region, and the third coding region are individually selected from the group consisting of a reporter sequence, a therapeutic gene, or signalling sequence, or a trafficking sequence.

35. The method of claim 34, wherein the first promoter sequence is a constitutive promoter sequence, the second promoter sequence is a tissue-specific promoter sequence, and the third promoter sequence is a lineage specific promoter sequence or a function-specific promoter sequence.

36. The method of claim 30, wherein the first promoter sequence, the second promoter sequence, and the third promoter sequence are selected from the group consisting of a constitutive promoter, a tissue-specific promoter sequence, and a lineage-specific promoter sequence.

37. The method of claim 1, wherein introducing the cell to the subject comprises intravenous administration, intracardiac administration, intradermal administration, intralesional administration, intrathecal administration, intracranial administration, intrapericardial administration, intraumbilical administration, intraocular administration, intraarterial administration, intraperitoneal administration, intraosseous administration, intrahemmorhage administration, intratrauma administration, intratumor administration, subcutaneous administration, intramuscular administration, intravitreous administration, direct injection into a normal organ, or direct injection into a diseased organ.

38. The method of claim 1, further comprising detecting expression of the first reporter by assaying for an association between the reporter expressed by the cell and the detectable moiety.

39. The method of claim 38, wherein the association between the cell and the detectable moiety comprises binding of the detectable moiety by the cell, binding of a ligand operably coupled to the detectable moiety by the cell, cellular uptake of the detectable moiety, or cellular uptake of a ligand operably coupled to the detectable moiety.

40. The method of claim 1, wherein the detectable moiety is a protein, a radioisotope, a fluorophore, a visible light emitting fluorophore, near infrared light emitting fluorophore, infrared light emitting fluorophore, a metal, a ferromagnetic substance, a paramagnetic substance, a superparamagnetic substance, a substance with a specific MR spectroscopic signature, an X-ray absorbing or reflecting substance, a sound altering substance, or an electromagnetic emitting substance.

41. The method of claim 40, wherein the detectable moiety is a radioisotope.

42. The method of claim 41 , wherein the detectable moiety is 111 -In octreotide.

43. The method of claim 39, wherein the detectable moiety is operably coupled to a ligand that specifically binds the reporter.

44. The method of claim 43, wherein the ligand is a nucleic acid such as a DNA or RNA molecule, a protein, a polypeptide, a peptide, an antibody, an antibody fragment, or a small molecule.

45. The method of claim 44, wherein the detectable moiety is a small molecule.

46. The method of claim 45, wherein contacting the cell with a detectable moiety occurs prior to introducing the cell to the subject.

47. The method of claim 45, further defined as comprising administering a detectable moiety to the subject after introducing the cell to the subject.

48. The method of claim 1, wherein the expression construct is comprised in a delivery vehicle, and wherein transferring into the cell an expression construct comprises contacting the cell with the delivery vehicle.

49. The method of claim 48, wherein said delivery vehicle is a lipid, a liposome, lipofectamine, a plasmid, a viral vector, a phage, a polyamino acid, a particle, calcium phoshate, DEAE-dextran, a procaryotic cell, or a eukaryotic cell.

50. The method of claim 49, wherein said delivery vehicle is a viral vector.

51. The method of claim 50, wherein said viral vector is a lentiviral vector, a baculovirus vector, a parvovirus vector, a semiliki forest virus vector, a Sindbis virus vector, a lentivirus vector, a retroviral vector, a vaccinia viral vector, an adeno-associated viral vector, a picornavirus vecctor, an alphavirus vector, or a poxviral vector.

52. The method of claim 51 , wherein said viral vector is a lentiviral vector.

53. The method of claim 1, wherein transferring the expression construct into the cell comprises performing electroporation or nucleofection of said cell in the presence of said expression construct.

54. The method of claim 1, wherein the imaging technique is an invasive imaging technique.

55. The method of claim 54a, wherein the invasive imaging technique comprises use of a catheter or endoscope.

56. The method of claim 1, wherein the imaging technique is a noninvasive imaging technique.

57. The method of claim 56, wherein the non-invasive imaging technique is selected from the group consisting of MRI, MR spectroscopy, radiography, CT, ultrasound, planar gamma camera imaging, SPECT, PET, other nuclear medicine-based imaging, optical imaging using visible light, optical imaging using luciferase, optical imaging using a fluorophore, other optical imaging, imaging using near infrared light, and imaging using infrared light.

58. The method of claim 1, further defined as a method of treating a subject with a disease.

59. The method of claim 58, wherein the disease is a hyperproliferative disease, an infectious disease, an inflammatory disease, a degenerative disease, a congenital disease, a genetic disease, an immunological disease, trauma, or a disease associated with toxicity or poisoning.

60. The method of claim 59, wherein the disease is a hyperproliferative disease.

61. The method of claim 60, wherein the disease is cancer.

62. The method of claim 58, wherein the disease is type I diabetes or type II diabetes.

63. The method of claim 58, wherein the disease is cardiovascular disease.

64. The method of claim 63, wherein the cardiovascular disease is cardiomyopathy, ischemic cardiac disease, infarction, congestive heart failure, congenital cardiac disease, traumatic cardiac disease, toxic cardiac disease, pericarditis, genetic cardiac disease.

65. The method of claim 58, wherein the disease is a neurological disease.

66. The method of claim 65, wherein the neurological disease is Parkinson's disease, Alzeimer disease, or multiple sclerosis.

67. The method of claim 58, wherein the cancer is breast cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, colon cancer, renal cancer, skin cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, uterine cancer, lymphatic cancer, stomach cancer, pancreatic cancer, testicular cancer, lymphoma, or leukemia.

68. The method of claim 58, wherein the diabetes is type I diabetes or type II diabetes.

69. The method of claim 58, wherein the neurological disease is neurodegenerative disease, spinal cord disease, traumatic neurological disease, infectious disease, or inflammatory disease,.

70. The method of claim 69, wherein the neurodegenerative disease is Parkinson's disease, Alzheimer disease, multiple sclerosis.

71. The method of claim 58, wherein the immunological disease is transplant rejection, autoimmune disease, immune complex disease, vasculitis, or HIV infection.

72. The method of claim 1, further defined as a method of assessing the viability of a stem cell in a subject.

73. The method of claim 1, further defined as a method of assessing the differentiation of a stem cell and/or its progeny in a subject.

74. The method of claim 1, further defined as a method for tracking the location of a tissue transplanted into a subject, wherein the cell is further defined as a cell comprised in a tissue, and wherein the tissue is transplanted into the subject.

75. The method of claim 1, wherein the first reporter further comprises a protein tag fused to the N-terminal end or C-terminal end of the truncated recombinant GPCR amino acid sequence.

76. The method of claim 75, wherein the protein tag has enzymatic activity.

77. The method of claim 76, wherein the protein tag is hemagglutinin A, beta- galactosidase, thymidine kinase, transferrin, myc-tag, VP 16, (His)6-tag, FLAG, or chloramphenicol acetyl transferase.

78. A method for tracking the location of a stem cell and/or its progeny in a human subject, comprising:

a) obtaining a stem cell;

b) transfecting the cell with an expression construct comprising a first coding region encoding a first reporter comprising somatostatin receptor truncated carboxy terminal to amino acid 314 and operatively linked to a first promoter sequence;

c) introducing the stem cell to the subject; and

d) detecting the location of the stem cell and/or its progeny in the subject using an imaging technique to detect a detectable moiety that is bound to the truncated SSTR2A.

79. A method for detecting the differentiation of a stem cell and/or its progeny in a human subject, comprising:

a) obtaining a stem cell;

b) transfecting the cell with an expression construct comprising a first coding region encoding a first reporter comprising a recombinant somatostatin receptor truncated carboxy terminal to amino acid 314 and operatively linked to a first promoter sequence;

c) introducing the stem cell to the subject; and

d) detecting the differentiation of the stem cell and/or its progeny in the subject using an imaging technique to detect a detectable moiety that is bound to the truncated recombinant somatostatin receptor.

80. The method of claim 78 or 79 wherein the stem cell is an immune progenitor cell, and wherein activation of the immune cell is detected.

81. A non-human transgenic animal whose genome comprises a nucleic acid encoding a truncated recombinant seven transmembrane G-protein associated receptor (GPCR) amino acid sequence.

82. The transgenic animal of claim 81, wherein the nucleic acid encoding a truncated recombinant seven transmembrane G-protein associated receptor (GPCR) amino acid sequence is operably linked to a promoter.

83. The transgenic animal of claim 81, wherein a cell of the transgenic animal expresses a truncated recombinant seven transmembrane G-protein associated receptor (GPCR) amino acid sequence.

84. The transgenic animal of claim 81, wherein the recombinant seven transmembrane G- protein associated receptor (GPCR) is a somatostatin receptor.

85. The transgenic animal of claim 81, wherein the somatostatin receptor is a somatostatin receptor type 2A (SSTR2A).

86. The transgenic animal of claim 85, wherein the somatostatin receptor is truncated carboxy terminal to amino acid 314.

87. The transgenic animal of claim 81, further comprising a protein tag fused to the N- terminal end or C-terminal end of the truncated recombinant GPCR amino acid sequence.

88. The transgenic animal of claim 87, wherein the protein tag has enzymatic activity.

89. The transgenic animal of claim 88, wherein the protein tag is hemagglutinin A, beta- galactosidase, thymidine kinase, transferrin, myc-tag, VP 16, (His)₆-tag, FLAG, or chloramphenicol acetyl transferase.

90. The transgenic animal of claim 82, wherein the promoter is a constitutive promoter, a tissue-selective promoter, a lineage-specific promoter sequence, or a functional promoter.

91. A non-human transgenic animal whose genome comprises a nucleic acid encoding a recombinant SSTR2 amino acid sequence, wherein the SSTR2 amino acid sequence is under the control of a heterologous promoter.

92. The transgenic animal of claim 91, wherein the promoter is a constitutive promoter, a tissue-selective promoter, a lineage-specific promoter sequence, or a functional promoter.

93. The transgenic animal of claim 91, further comprising a protein tag fused to the N- terminal end or C-terminal end of the truncated recombinant GPCR amino acid sequence.

94. The transgenic animal of claim 93, wherein the protein tag has enzymatic activity.

95. The transgenic animal of claim 94, wherein the protein tag is hemagglutinin A, beta- galactosidase, thymidine kinase, transferrin, myc-tag, VP 16, (His)₆-tag, FLAG, or chloramphenicol acetyl transferase.

96. A method of producing a cell that expresses a truncated recombinant seven transmembrane G-protein associated receptor (GPCR), comprising obtaining the transgenic animal of claim 81 and isolating one or more cells from said transgenic animal.

97. The method of claim 96, wherein the cell is a stem cell, an immune cell, or a cancer cell.

98. The method of claim 97, wherein the stem cell is an embryonic stem cell or a somatic stem cell.

99. The method of claim 96, wherein the recombinant seven transmembrane G-protein associated receptor (GPCR) is a somatostatin receptor.

100. The method of claim 99, wherein the somatostatin receptor is a somatostatin receptor type 2A (SSTR2A).

101. The method of claim 99, wherein the somatostatin receptor is truncated carboxy terminal to amino acid 314.

102. The method of claim 96, further comprising sorting the one or more cells that have been isolated.

103. The method of claim 102, wherein sorting the cells comprises performing or more techniques selected from the group consisting of FACS, separation using magnetic resonance beads, and column chromatography.

104. A method of producing a cell and/or progeny of a cell that expresses a somatostatin receptor amino acid sequence, comprising (a) obtaining a non-human transgenic animal whose genome comprises a nucleic acid encoding a first reporter comprising a somatostatin receptor amino acid sequence and (b) isolating one or more cells or tissues from said transgenic animal.

105. The method of claim 104, wherein the somatostatin receptor amino acid sequence is a somatostatin receptor type 2A (SSTR2A) amino acid sequence.

106. A method for tracking the location of a cell and/or progeny of a cell in a subject, comprising:

a) contacting the cell and/or progeny of the cell produced by the method of claim 104 or claim 105 with a detectable moiety that binds to the first reporter;

b) introducing the cell and/or progeny of the cell to the subject; and

c) imaging the detectable moiety using an imaging technique.

107. The method of claim 106, further defined as a method of treating a subject with a disease.

108. The method of claim 106, wherein the disease is a hyperproliferative disease, an infectious disease, an inflammatory disease, a degenerative disease, a congenital disease, a genetic disease, an immunological disease, trauma, a neurological disease, or a disease associated with toxicity or poisoning.

109. The method of claim 108, wherein the disease is a hyperproliferative disease that is cancer.

110. The method of claim 109, wherein the cancer is breast cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, colon cancer, renal cancer, skin cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, uterine cancer, lymphatic cancer, stomach cancer, pancreatic cancer, testicular cancer, lymphoma, or leukemia.

111. The method of claim 107, wherein the disease is type I diabetes or type II diabetes.

112. The method of claim 107, wherein the disease is cardiovascular disease.

113. The method of claim 112, wherein the cardiovascular disease is cardiomyopathy, ischemic cardiac disease, infarction, congestive heart failure, congenital cardiac disease, traumatic cardiac disease, toxic cardiac disease, pericarditis, genetic cardiac disease.

114. The method of claim 108, wherein the neurological disease is Parkinson's disease, Alzeimer disease, multiple sclerosis, neurodegenerative disease, spinal cord disease, traumatic neurological disease, infectious disease, or inflammatory disease.

115. The method of claim 108, wherein the immunological disease is transplant rejection, autoimmune disease, immune complex disease, vasculitis, or HIV infection.