WO2002003789A2

WO2002003789A2 - Transgenic mice containing targeted gpcr gene disruptions

Info

Publication number: WO2002003789A2
Application number: PCT/US2001/021498
Authority: WO
Inventors: Keith D. Allen; Thomas J. Brennan
Original assignee: Deltagen Inc
Current assignee: Deltagen Inc
Priority date: 2000-07-06
Filing date: 2001-07-06
Publication date: 2002-01-17
Anticipated expiration: 2003-01-06
Also published as: WO2002003789A3; AU2001271902A1

Abstract

The present invention relates to transgenic animals, as well as compositions and methods relating to the characterization of gene function. Specifically, the present invention provides transgenic mice comprising mutations in a target GPCR gene. Such transgenic mice are useful as models for desease and for identifying agents that modulate gene expression and gene function, and as potential treatments for various disease states and disease conditions.

Description

TRANSGENIC MICE CONTAINING TARGETED GPCR GENE DISRUPTIONS

Field of the Invention

The present invention relates to transgenic animals, compositions and methods relating to the characterization of gene function. Background of the Invention

Experimental animal models are important tools for understanding the role of genes. More particularly, the ability to develop animals with specific genes altered or inactivated has been invaluable to the study of gene function, and has lead to unexpected discoveries of a gene and/or mechanisms responsible for disease with similar manifestations in humans. These genetically engineered animals are also useful for identifying and testing therapeutic treatments for a variety of diseases and disorders.

The identification of the function of numerous genes has been useful in ascertaining the roles of these genes in disease. Because of the high level of homology between humans and mice, for example, it is possible to define the function of individual human genes by making targeted germline mutations in selected genes in the animal. The phenotype of the resulting mutant animal can be used to help define the phenotype in humans.

Several interesting genes have recently been discovered belonging to various families encoding G-protein coupled receptors (GPCRs), LDL receptors, cerberus and cerberus-like proteins, and proteins involved in carbohydrate metabolism, particularly those involved in epithelial develop- ment. Identifying the roles of these genes and their expression products may permit the definition of disease pathways and the identification of diagnostically and therapeutically useful targets.

GPCRs have been characterized as having seven putative transmembrane domains (designated TM1, TM2, TM3, TM4, TM5, TM6, and TM7) which are believed to represent transmembrane α-helices connected by extracellular or cytoplasmic loops. Most G-protein coupled receptors have single conserved cysteine residues in each of the first two extracellular loops which form disulfide bonds that are believed to stabilize functional protein structure. G-protein coupled receptors can be intracellularly coupled by heterotrimeric G-proteins to various intracellular enzymes, ion channels and transporters. Different G-protein -subunits preferentially stimulate particular effectors to modulate various biological functions in a cell. One important subfamily of GPCRs is the chemokine or chemokine-like receptors. Recently, a novel orphan GPCR termed the DEZ receptor (aka chemokine-like receptor 1, or CMKLRl) was isolated from a cDNA derived from the cell line NH15-CA2 and a cDNA library from adult mouse brain using a PCR cloning strategy (Methner et al, Biochem. Biophys. Res. Commun. 233(2): 336-342 (1997)). The amino acid sequence of this DEZ orphan receptor showed homology to neuropeptide and chemoattractant receptors. Highest overall homology was found with the orphan receptor GPR-1 (65%), the angiotensin II receptor (62%), and the C5a anaphylatoxin receptor (60%). In situ hybridization experiments showed that dez is differentially regulated during development, with a prominent expression in developing osseous and cartilaginous tissue. It was also detectable in the adult parathyroid glands, hinting at a possible function in bone metabolism. However, the structural and functional attributes of this receptor have yet to be determined. The complete 1892 bp cDNA eds for the murine DEZ orphan receptor, or CMKLRl, has been deposited in GenBank (Accession number: U79525; GIZMO number: 1732346).

Another important subfamily of GPCRs is the cannabinoid receptors. The peripheral cannabinoid receptor, mCB2, from a mouse splenocyte cDNA library, was recently cloned (Biochim. Biophys. Acta 1307(2), 132-6 (1996)). The 3715 bp sequence contains an open reading frame encoding a protein of 347 residues sharing 82% overall identity with the only other known peripheral receptor, human CB2 (hCB2) and shorter than hCB2 by 13 amino acids at the carboxyl terminus. Binding experiments with membranes from COS-3 cells transiently expressing mCB2 showed that the synthetic cannabinoid WIN 55212-2 had a 6-fold lower affinity for mCB2 than for hCB2, whereas both receptors showed similar affinities for the agonists CP 55,940, delta(9)-THC and anandamide and almost no affinity for the central receptor-(CB 1) specific antagonist SR 141716A. Both hCB2 and mCB2 mediate agonist-stimulated inhibition of forskolin-induced cAMP production in CHO cell lines permanently expressing the receptors. SR 141716A failed to antagonize this activity in either cell line, confirming its specificity for CB1. The full-length 3715 bp cDNA for the murine CB2 receptor (CB2R) gene sequence has been submitted to GenBank (GI or NID number: 791081; Accession number: X86405). The CB2R coding sequence is believed to comprise bases 116-1159.

An important member of the GPCRs is the adrenomedullin receptor. Adrenomedullin, a hypotensive peptide found in human pheochromocytoma, consists of 52 amino acids, has one intramolecular disulfide bond, and shows a slight homology with the calcitonin gene-related peptide. Adrenomedullin is a potent vasodilator peptide that exerts major effects on cardiovascular function. Its actions are mediated through an abundant class of specific binding sites that activate adenylyl cyclase through a G protein-coupled mechanism. Adrenomedullin has recently been associated with human tumor growth (J. Biol. Chem. 271, 23345-23351 (1996)). A partial cDNA eds (526 bp) for the murine adrenomedullin receptor gene that is abundantly expressed in liver and lung has been submitted to GenBank (GI or NTD number: 2150047; Accession number: AF001229). Another important subfamily of GPCRs is the corticotropin-releasing factor receptor (CRFR), or corticotropin-releasing hormone receptor (CRHR), GPCRs. Corticotropin-releasing factor (CRF), aka corticotropin-releasing hormone, is a 41 -residue hypothalamic peptide which stimulates the secretion and biosynthesis of pituitary ACTH, leading to increased adrenal glucocorticoid production. CRF was originally isolated and characterized on the basis of its role in the hypothalamic -pituitary- adrenal axis (HPA) Science 213, 1394-1397 (1981)). More recently, however, it has been found to be distributed broadly within the central nervous system (CNS), as well as in extra-neural tissues such as the adrenal glands and testes (Neuroendocrinology 36, 165-186 (1983); J. Clin. Endocrinol. Metab. 58, 919-924 (1984); Endocrinology 127, 1541-1543 (1990)), where it may also act as a paracrine regulator or neurotransmitter.

In addition to its critical role of mediating HPA axis activation, CRF has been shown to modulate behavioral changes that occur during stress response. Many of these behavioral changes have been shown to occur independently of HPA activation in that they are insensitive to dexamethasone treatment and hypophysectomy (Life Sci. 38, 211-216 (1986); Life Sci. 39, 1281-1286 (1986); Pharm. Bioch. Behav. 34, 517-519 (1989)). In addition, direct infusion of CRF into the CNS mimics autonomic and behavioral responses to a variety of stressors (Nature 297, 331-333 (1982); Brain Res. 280, 75-79 (1983); Peptides 9, 1067-1070 (1988); J. Neurosci. 10, 176-183 (1990)).

Central administration of CRF in rodent animal models produces effects that correlate with a state of anxiety, such as a reduction in willingness to investigate unfamiliar surroundings (Pharm. Biochem. Behav. 26, 699-703 (1987)), decreased sleep, enhanced fear responses, decreased food consumption (Life Sci. 31, 1459-1464 (1982)) and suppressed sexual behavior (Nature 305, 232-235 (1983)). These changes are similar to behavioral changes observed upon exposure to acute and chronic stressors, and resemble changes that occur in human affective disorders such as the symptom complex characteristic of major depressive disorder, panic disorder and anorexia nervosa (J. Clin. Endocrinol. Metab. 64, 203-208 (1987); N. Engl. J. Med. 319, 413-420 (1988); Psych. Clin. N. Amer. 22, 335-348 (1988); Pharmacpsychiat. 21, 76-82 (1988)). Currently, a great deal of interest in CRF has been generated in connection with the pathophysiology of mental illness. For example, CRF hypersecretion has been linked to some individuals diagnosed with major depression (Science 226, 1342-1344 (1984)). Indeed, a large portion of individuals diagnosed with depression have elevated cortisol levels (Am. J. Psychiatry 143, 896-899 (1987); /. Clin. Endocrinol. & Metab. 72, 260-271 (1991)). Moreover, in major depression (N. Engl. J. Med. 311, 1127 (1984)) and panic disorder, CRF administration results in a blunted ACTH response, suggesting that the pituitary is properly restrained, presumably by the negative feedback effect of elevated levels of glucocorticoids. In view of these findings, it has been suggested that the hypercortisolism in major depression is due to abnormal CRF secretion within the CΝS (Psychiat. Clinics ofN. Amer. 11, 327-335 (1988)). Liaw et al. (1996), Endocrinology 137: 72-77, reported that there are two G protein-coupled

CRH receptors, CRHRl and CRHR2, the latter of which they termed the CRF2 receptor (or CRFR2). The investigators used clones of the rat CRF2 receptor to isolate the human gene from brain and kidney genomic DΝA libraries. The gene consists of 12 exons spanning approximately 30 kb. The predicted protein, which is 411 amino acids in length and 70% identical to the CRFl receptor, contains a putative Ν-terminal secretory signal sequence and 7 putative transmembrane domains. Liaw et al. (1996) expressed the CRF2 receptor and found that transfected cells responded to the binding of CRH with an increase in intracellular cAMP. Although the rat receptor has two alternatively spliced variants, termed CRF2-alpha and CRF2-beta, Liaw et al. (1996) found no evidence for alternative splicing of the human receptor. Liaw et al. (1996) reported that the pharmacologic profile of this protein was similar to that of the rat CRF2-alpha protein but distinct from the human CRFl receptor.

Meyer et al. (1997), Genomics 40: 189-190, used radiation hybrids and fluorescence in situ hybridization to map CRHR2 to human chromosome 7p21-ρl5 between the STS markers D7S632 and D7S690. Hsu and Hsueh (2001), Nature Med. 7: 605-611, have identified stresscopin (SCP) and stresscopin-related peptide (SRP) as specific ligands for CRHR2. Kostich et al. (1998), Molec. Endocr. 12: 1077-1085, reported a novel CRHR2 splice isoform, which they referred to as 'CRF2-gamma,' found in human brain. CRF2-gamma cDNA encodes a 397- amino acid receptor containing an amino terminus with no significant homology to the already reported alpha- and beta-termini. PCR and Southern blot analysis of CRF2-gamma RNA expression in human brain detected expression in the septum and hippocampus, with weaker but detectable expression in the amygdala, nucleus accumbens, midbrain, and frontal cortex.

Two CRF receptors, CRFR1 and CRFR2, have been identified in mice. While CRFRl shares 70% sequence identity with CRFR2, it has much higher affinity for rat/human CRF. Lesh et al. (1997), Mammalian Genome 8: 944-945, mapped the mouse CRFR2 gene to chromosome 6.

A murine gene encoding a CRFR expressed primarily in heart and skeletal muscle (i.e., the heart/muscle HM-CRFR) has been isolated and characterized (Proc. Natl. Acad. Sci. USA 92(A), 1108-12 (1995), the disclosure of which is incorporated herein by reference). The complete cDNA eds (1557 bp) bears GenBank GI or NID number 717137 and Accession number U21729. The coding sequence is believed to comprise bases 127-1422. The murine HM-CRFR gene corresponds with murine CRFR2 (aka CRHR2) gene. Coste et al. (2000), Nature Genet. 24: 403-409, generated CRHR2 -/- mice through targeted disruption and demonstrated that CRHR2 supplies regulatory features of the hypothalamic-pituitary- adrenal axis stress response. Although initiation of the stress response appeared to be normal, CRHR2 -/- mice showed early termination of adrenocorticotropic hormone (Acth) release, suggesting that CRHR2 is involved in maintaining hypothalamic-pituitary-adrenal axis drive. CRHR2 also appears to modify the recovery phase of the hypothalamic-pituitary-adrenal axis response, as corticosterone levels remained elevated after stress in CRHR2 -/- mice. In addition, stress coping behaviors associated with dearousal were reduced in CRHR2 -/- mice. Coste et al. (2000) also demonstrated that CRHR2 is essential for sustained feeding suppression induced by urocortin. Feeding was initially suppressed in CRHR2 -/- mice following urocortin administration, but CRHR2 -/- mice recovered more rapidly and completely than did wildtype mice. In addition to central nervous system effects, Coste et al. (2000) found that, in contrast to wildtype mice, CRHR2 -/- mice failed to show the enhanced cardiac performance or reduced blood pressure associated with systemic urocortin, suggesting that CRHR2 mediates these peripheral human dynamic effects. Moreover, CRHR2 -/- mice have elevated basal blood pressure, demonstrating that CRHR2 participates in cardiovascular homeostasis. Bale et al. (2000), Nature Genet. 24: 410-414, generated CRHR2 -/- mice by targeted disruption. They found that CRHR2 -/- mice were hypersensitive to stress and displayed increased anxiety-like behavior. Mutant mice had normal basal feeding and weight gain but decreased food intake following food deprivation. Intravenously injected urocortin produced no effect on mean arterial pressure in the mutant mice. Kishimoto et al. (2000), Nature Genet. 24: 415-419, generated mice deficient for CRHR2 by targeted disruption. They reported that male but not female CRHR2-deficient mice exhibited enhanced anxious behavior in several tests of anxiety in contrast to mice lacking CRHRl. The enhanced anxiety of CRHR2-deficient mice was not due to changes in hypothalamic-pituitary-adrenal axis activity, but rather reflected impaired responses in specific brain regions involved in emotional and autonomic functions, as monitored by a reduction in Creb phosphorylation in male, but not female, CRHR2 -/- mice. Kishimoto et al. (2000) proposed that CRHRl predominantly mediates a central anxiolytic response, opposing the general anxiogenic effect of CRH mediated by CRHRl. Kishimoto et al. (2000) found that neither male nor female CRHR2-deficient mice showed alterations of baseline feeding behavior. Both responded with increased edema formation in response to thermal exposure, however, indicating that in contrast to its central role in anxiety, the peripheral role of CRHR2 in vascular permeability is independent of gender.

Another important subfamily of GPCRs is the N-formylpeptide receptor GPCRs. Control of formylpeptide chemoattractant receptors is a process of central importance to chemotaxis of phagocytes towards sites of bacterial infection. Polymorphonuclear neutrophils are attracted to sites of infection by a variety of specific substances including a number of N-formylmethionyl peptides. The N-formylated peptides are believed to derive from bacterial protein degradation or from mitochondrial proteins upon tissue damage. Activation of the N-formylpeptide receptor (FPR) of human neutrophils by ligands such as N-formyl-methionine-leucine-phenylalanine (/MLP) induces mobilization of intracellular calcium, cell adhesion, chemotaxis, superoxide production and degranulation, as important components of the inflammatory response. Stimulation of the formyl peptide receptor activates a phospholipase C which in turn results in production of the intracellular second messengers diacylglycerol and inositol triphosphate. These second messengers activate the protein kinase C and mobilize calcium from intracellular stores, respectively. Boulay et al. (Biochemistry 29:11123-11133 (1990)). characterized two cDΝA isolates for the Ν-formylpeptide receptor. Sequence comparisons established that the Ν-formylpeptide receptor belongs to the GPCR superfamily. The membrane skeleton meshwork (MSK) underlying the plasma membrane of human neutrophils has been implicated in the regulation of these receptors. The association of FPR with the MSK, measured by pelletablility of FPR in Triton X-100 extracts of membranes, depends on the stimulation state of the cell, showing minimal association in membranes from unstimulated cells and nearly complete association in membranes from homologously desensitized cells. If membranes are more completely solubilized in octyl glucoside and then exchanged into Triton X-100, FPR is found in tight association with a protein immunologically related to actin.

Recently, a murine N-formylpeptide receptor-like 3 (FPRL3 or FPR-RS3) gene has been reported (Genomics 51(2):270-276 (1998)). A 1444 bp eds sequence for the murine FPRL3 gene has been submitted to GenBank (GI or MD number: 3549281; Accession number: AF071181). The coding sequence is believed to comprise bases 224-1255.

Knockout mice lacking the high affinity N-formylpeptide receptor type 1 (FPR1) have been created by targeted gene disruption . (J. Exp. Med. 189(4):657-62 (1999)). FPR1 -/- mice developed normally, but had increased susceptibility to challenge with Listeria monocytogenes, as measured by increased mortality compared with wild-type littermates. FPRl -/- mice also had increased bacterial load in spleen and liver two days after infection, which is before development of a specific cellular immune response, suggesting a defect in innate immunity. Consistent with this, neutrophil chemotaxis in vitro and neutrophil mobilization into peripheral blood in vivo in response to the prototype N- formylpeptide fMLF (foπnyl-methionyl-leucyl-phenylalanine) were both absent in FPRl -/- mice. These results indicate that FPRl functions in antibacterial host defense in vivo.

Another important subfamily of GPCRs is the neuropeptide Y receptor GPCRs. Neuropeptides are small peptides originating from large precursor proteins synthesized by peptidergic neurons and endocrine/paracrine cells, and hold promise for treatment of neurological, psychiatric, and endocrine disorders. Often, the precursors contain multiple biologically active peptides. There is great diversity of neuropeptides in the brain caused by alternative splicing of primary gene transcripts and differential precursor processing. The neuropeptide receptors serve to discriminate between ligands and to activate the appropriate signals.

Neuropeptide Y (NPY), the most abundant neuropeptide to be identified in mammalian brain, is a 36 amino acid peptide which belongs to a family of neuroendocrine peptides consisting of NPY, peptide YY (PYY) and pancreatic polypeptide (PP). It is widely distributed throughout the central and peripheral nervous systems of mammals (Gen. Pharmacol. 24, 785 (1993); Cardiovasc. Res. 27, 893 (1993)). In the brain, NPY is particularly abundant in the hypothalamus, the limbic system and the cortex (Neurosci. 15, 1149 (1985)). In the periphery, it is localized in sympathetic nerve fibers that surround blood vessels and other smooth muscle tissues. NPY exerts a remarkably wide variety of physiological effects of potential therapeutic importance. It induces vasoconstriction when administered alone and acts synergistically when administered with other vasoconstrictors such as KC1, ATP, angiotensin II and histamine (Ann. NY Acad. Sci. 611, 7 (1990); Ann. NY Acad. Sci. 611, 166 (1990); Am. J. Physiol. 258, R736 (1990); and Gen. Pharmacol. 20, 363 (1989)). When acting upon coronary arteries, the vasoconstrictive action of NPY can cause angina pectoris (Lancet 1(8541), 1057 (1987)). In addition, NPY has been found to restore the response to vasoconstrictors after desensitization which follows multiple exposure to vasoactive substances or after endotoxic shock (Am. J. Physiol. 265, H1416 (1993)). NPY also exerts a mitogenic effect on aortic and venous smooth muscle tissue, and may contribute to cardiovascular hypertrophy in hypertension. Recent data suggests that it may promote angiogenesis as efficiently as basic fibroblast growth factor (Peptides IA, 263 (1993)). A third physiological action of NPY is in the hypothalamic regulation of body temperature, energy balance and metabolism. There is a large body of evidence indicating that NPY induces food intake in animals when injected in the hypothalamic area (Endocrinol. 115, 427 (1984); Life Sci. 35, 2635 (1984); Peptides 5, 1025 (1984)). Recent reports suggest that it is the major mediator of the action of OB/leptin, a protein which acts centrally to reduce food consumption (Nature 377, 530 (1995)), and that it has a direct anti-lipolytic effect on adipocytes (Am. J. Physiol. 265, E74 (1993)). Other results suggest that NPY may be important in the treatment of some forms of type II diabetes (Diabetes 40, 660 (1991); Regul. Peptides 34, 225 (1991)). Intracerebroventricular injection of the peptide enhances insulin secretion from pancreatic islets via autonomic control, whereas, in the periphery, NPY has a direct inhibitory effect on pancreatic insulin release. Still another physiological effect reported for NPY is in the regulation of gonadotropin secretion. There are indications that it may play a role in follicular maturation and ovulation (Biochem. Biophys. Res. Comm. 200, 1111 (1994); Ann. NY Acad. Sci. 611, 273 (1990); Neuropep. 30, 293 (1996)). It has been found that NPY levels are elevated in rats with decreased sexual function and that ventral administration of the peptide reduces sexual performance. In addition, several lines of evidence indicate that sex steroids exert a feedback regulation on NPY levels (Endocrinol. 130, 3331 (1992); Endocrinol. 132, 139 (1993)). Still other effects of NPY include: improved memory retention as observed in mice (/. Pharmacol. Exp. Tlier. 268, 1010 (1994)); analgesia in animal models of pain (Brain Res. 724, 25 (1996)); inhibition of the excitatory amino acid glutamate, suggesting a possible role in epilepsy (Br. J. Pharmacol. 113, 737 (1994)); and modulation of nasal vasodilation, rhinorrhea and bronchial secretion, suggesting possible importance in treating allergic rhinitis and cystic fibrosis (Br. J. Pharmacol. 118, 2079 (1996); Am. J. Physiol. 266, L513 (1994)).

Reduced cortical concentrations of NPY have been observed in animal models of depression, and antidepressants have been found to increase NPY production (Clin. Neurophannacol. 9(4), 572 (1986)). NPY has been reported to produce an anxiolytic effect in animal models of anxiety (Psychopharmacol. 98, 524 (1989)). In addition, concentrations of NPY are reduced in the CSF of patients with major depression or severe anxiety and in the brain tissue of some suicide victims (Annu. Rev. Pharmacol. Toxicol. 32, 309 (1993)). At least six distinct subtypes of NPY receptors have been described. The receptor subtypes, named Yl, Y2, etc., were initially classified based upon their selectivity for NPY, PYY and PP, as well as for their binding to NPY analogues and C-terminal fragments.

Recently, the complete eds of a murine pancreatic neuropeptide Y receptor gene, NPY4-R (aka NPYR-D), has been reported (FEBS Lett. 381(1-2), 58-62 (1996)). The NPY4-R sequence (GI or D number: 1223969; Accession number: U40189) comprises 1500 bp and is believed to encode an intronless receptor comprising 375 amino acids. Percent identities of NPY4-R to the clone Yl, Y2, rat Y4/PP1 and human Y4 PP1 receptors were found to be 45%, 32%, 92% and 76%, respectively. Southern blots indicate that NPY4-R and human Y4 PP1 receptor genes are species homologues. Tissue distribution studies in mouse and humans suggested potential roles of this receptor in the gastrointestinal tract, heart, prostate, as well as in neural and endocrine signaling. More recently, a transgenic mouse with a disrupted NPY-Y1 gene has been reported (U.S. Patent No. 5,817,912); this

"knockout" mouse was studied with respect to eating behavior and weight changes.

Recently, the complete eds of a murine pancreatic neuropeptide Y receptor gene, NPY6-R (aka pancreatic polypeptide receptor 2, or PP2), has been reported to be cloned and expressed from mouse genomic DNA (J. Biol. Chem. 271(28): 16435-8 (1996)). In situ hybridization of mouse brain sections revealed expression of this receptor within discrete regions of the hypothalamus including the suprachiasmatic nucleus, anterior hypothalamus, bed nucleus stria terminalis, and the ventromedial nucleus with no localization apparent elsewhere in the brain. The NPY6-R gene sequence (GenBank GI or NID number: 1378003; Accession number: U58367) comprises 2281 bp; the coding region is believed to comprise bases 823-1938.

Another important subfamily of GPCRs is the opiate receptor GPCRs. Opiates are a large class of neurotransmitters and substances, mediating various behaviors and responses such as analgesia and euphoria. In clinical settings, these compounds are used for pain management, but often have undesirable side effects. The diverse activities of various opiates led to the initial suggestion that there were divergent receptors for these compounds. An opiate GPCR receptor from mouse brain, kappa-3 (or K3 opiate receptor, or KOR-3, or KOR3), has recently been identified, which is distinct from the previously cloned mu, delta, and kappa 1 receptors; assignment of the clone to the opiate receptor subfamily was derived from both structural and functional studies (Mol. Pharmacol. Al (6), 1180-1188 (1995)). The complete cDNA eds for the murine KOR3 gene, comprising 2600 bp, is known (GenBank GI or NID Number: 551484; Accession Number: U09421); bases 299-1402 are believed to comprise the coding sequence.

Another important subfamily of GPCRs is the metabotropic glutamate receptors. The majority of nerve cell connections are chemical synapses. A neurotransmitter is released from the presynaptic terminal, typically in response to the arrival of an action potential in the neuron, and diffuses across the synaptic space to bind to membrane receptor proteins of the postsynaptic terminal. The binding of neurotransmitters to membrane receptors is coupled either to the generation of a permeability change in the postsynaptic cell or to metabolic changes. Neurotransmitters produce different effects according to the type of receptor to which they bind. In general, those which produce effects that are rapid in onset and brief in duration bind receptors that act as ligand-gated ion channels, where binding almost instantly causes an ion flow across the membrane of the postsynaptic cell. Those neurotransmitters that act more like local chemical mediators bind to receptors that are coupled to intracellular enzymes, thereby producing effects that are slow in onset and more prolonged. These neurotransmitters alter the concentration of intracellular second messengers in the postsynaptic cell. The ligand-activated membrane receptors do not activate the second messenger systems directly, but rather via membrane-bound G proteins, which bind GTP on the cytoplasmic surface of the cell membrane. Glutamate^', aspartate and their endogenous derivatives are believed to be the predominant excitatory neurotransmitters in the vertebrate central nervous system. Recently, glutamate has been described as playing a major role in the control of neuroendocrine neurons, possibly controlling not only the neuroendocrine system but other hypothalamic regions as well. Four major subclasses of glutamate receptors have been described. Three of the receptors, the quisqualate (QA/AMPA), kainate (KA), and N-methyl-D-aspartate (NMDA) receptors, are believed to be directly coupled to cation-specific ion channels and thus are classified as ligand-gated ionotropic receptors. The fourth glutamate receptor binds some of the agonists of the ionotropic receptors (quisqualate and glutamate, but not AMPA) but has no shared antagonists, and is coupled to G protein. Thus, this receptor, referred to as the G protein-coupled glutamate receptor is pharmacologically and functionally distinct from the other major glutamate receptors. This receptor has also been termed the metabotropic glutamate receptor.

The metabotropic glutamate receptors (mGluRs) have been divided into three groups on the basis of sequence homology, putative signal transduction mechanisms, and pharmacologic properties. Group II and group ILT mGluRs are linked to the inhibition of the cyclic AMP cascade, but differ in their agonist selectivities. The cDNAs encoding 3 human group IH mGluRs, mGluR4, mGluR7 and mGluR8, have been isolated. The predicted 908-amino acid human mGluR8 protein shares 67 to 70% sequence similarity with mGluR4 and mGluR7 and 99% amino acid identity with mouse mGluR8. Recently, a novel metabotropic glutamate receptor, mGluR8, was identified by screening a mouse retina cDNA library (/. Neurosci. 15(4), 3075-83 (1995)). In situ hybridization studies revealed a strong expression of the mGluR8 gene in the olfactory bulb, accessory olfactory bulb, and mammillary body. A weaker expression was found in the retina, and in scattered cells in the cortex and hindbrain. The pharmacology and expression of mGluR8 in mitral/tufted cells suggest it could be a presynaptic receptor modulating glutamate release by these cells in their axon terminals in the entorhinal cortex. The full-length cDNA eds for the murine mGluR8 (aka MGR8) gene (GenBank GI/NID Number: 854728; Accession Number: U17252) consists of 2830 bp, wherein bases 71-2797 are believed to comprise the coding sequence.

Another important member of the GPCRs is the mas oncogene. The human mas (aka MAS1) oncogene was isolated from the DNA of a human epidermoid carcinoma cell line and, based on its deduced amino acid sequence, the MAS1 gene product contains 7 potential transmembrane domains. The masl protein is, therefore, probably an integral membrane protein. The masl encoded protein is believed to be a receptor that, when activated, modulates a critical component in a growth-regulating pathway to bring about its oncogenic effects.

Recently, the mas proto-oncogene was isolated from a mouse genomic library (FEBS Lett. 357(1), 27-32 (1995)). Sequence analysis showed that it contained an open reading frame without intervening sequences. The amino acid sequence deduced confirmed the seven-transmembrane- domain structure and exhibited 97% and 91% amino acid homology with the rat and the human Mas, respectively. In mice and rats, mas mRNA was detected in the testis, kidney, heart, and in the brain regions: hippocampus, forebrain, piriform cortex, and olfactory bulb. The product of the mouse MAS gene is believed to be involved in the development of the brain and testis. The full-length cDNA for the murine MAS gene consists of 1216 bp and bears GenBank GI or NID number 53011 and Accession number X67735. The coding sequence for the murine MAS gene is believed to comprise bases 147-1121.

Summary of the Invention The present invention generally relates to transgenic animals, as well as to compositions and methods relating to the characterization of gene function.

The present invention provides transgenic cells comprising a disruption in a targeted gene. The transgenic cells of the present invention are comprised of any cells capable of undergoing homologous recombination. Preferably, the cells of the present invention are stem cells and more preferably, embryonic stem (ES) cells, and most preferably, murine ES cells. According to one embodiment, the transgenic cells are produced by introducing a targeting construct into a stem cell to produce a homologous recombinant, resulting in a mutation of the targeted gene. In another embodiment, the transgenic cells are derived from the transgenic animals described below. The cells derived from the transgenic animals includes cells that are isolated or present in a tissue or organ, and any cell lines or any progeny thereof.

The present invention also provides a targeting construct and methods of producing the targeting construct that when introduced into stem cells produces a homologous recombinant. In one embodiment, the targeting construct of the present invention comprises first and second polynucleotide sequences that are homologous to the targeted gene. The targeting construct also comprises a polynucleotide sequence that encodes a selectable marker that is preferably positioned between the two different homologous polynucleotide sequences in the construct. The targeting construct may also comprise other regulatory elements that may enhance homologous recombination.

The present invention further provides non-human transgenic animals and methods of producing such non-human transgenic animals comprising a disruption in a target gene. The transgenic animals of the present invention include transgenic animals that are heterozygous and homozygous for a mutation in the target gene. In one aspect, the transgenic animals of the present invention are defective in the function of the target gene. In another aspect, the transgenic animals of the present invention comprise a phenotype associated with having a mutation in a target gene.

The present invention also provides methods of identifying agents capable of affecting a phenotype associated with a disruption of a target gene in a transgenic animal. For example, a putative agent is administered to the transgenic animal and a response of the transgenic animal to the putative agent is measured and compared to the response of a "normal" or wild type mouse, or alternatively compared to a transgenic animal control (without agent administration). The invention further provides agents identified according to such methods. The present invention also provides methods of identifying agents useful as therapeutic agents for treating conditions associated with a disruption of the target gene.

The present invention further provides a method of identifying agents having an effect on target gene expression or function. The method includes administering an effective amount of the agent to a transgenic animal, preferably a mouse. The method includes measuring a response of the transgenic animal, for example, to the agent, and comparing the response of the transgenic animal to a control animal, which may be, for example, a wild-type animal or alternatively, a transgenic animal control. Compounds that may have an effect on target gene expression or function may also be screened against cells in cell-based assays, for example, to identify such compounds.

The invention also provides cell lines comprising nucleic acid sequences of a target gene. Such cell lines may be capable of expressing such sequences by virtue of operable linkage to a promoter functional in the cell line. Preferably, expression of the target gene sequence is under the control of an inducible promoter. Also provided are methods of identifying agents that interact with the target gene, comprising the steps of contacting the target gene or target protein with an agent and detecting an agent/ target gene or agent/target protein complex. Such complexes can be detected by, for example, measuring expression of an operably linked detectable marker.

The invention further provides methods of treating diseases or conditions associated with a disruption in a target gene, and more particularly, to a disruption in the expression or function of the target gene or the target protein encoded by the target gene. In a preferred embodiment, methods of the present invention involve treating diseases or conditions associated with a disruption in the target gene's expression or function, including administering to a subject in need, a therapeutic agent which effects target gene or target protein expression or function. In accordance with this embodiment, the method comprises administration of a therapeutically effective amount of a natural, synthetic, semi- synthetic, or recombinant target gene, target gene products or fragments thereof as well as natural, synthetic, semi-synthetic or recombinant analogs.

The present invention further provides methods of treating diseases or conditions associated with disrupted targeted gene expression or function, wherein the methods comprise detecting and replacing through gene therapy mutated target genes. Definitions

The term "gene" refers to (a) a gene containing at least one of the DNA sequences disclosed herein; (b) any DNA sequence that encodes the amino acid sequence encoded by the DNA sequences disclosed herein and/or; (c) any DNA sequence that hybridizes to the complement of the coding sequences disclosed herein. Preferably, the term includes coding as well as noncoding regions, and preferably includes all sequences necessary for normal gene expression including promoters, enhancers and other regulatory sequences.

The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably to refer to polymeric forms of nucleotides of any length. The polynucleotides may contain deoxyribonucleotides, ribonucleotides and/or their analogs. Nucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The term "polynucleotide" includes single-, double-stranded and triple helical molecules.

"Oligonucleotide" refers to polynucleotides of between 5 and about 100 nucleotides of single- or double-stranded DNA. Oligonucleotides are also known as oligomers or oligos and may be isolated from genes, or chemically synthesized by methods known in the art. A "primer" refers to an oligonucleotide, usually single-stranded, that provides a 3'-hydroxyl end for the initiation of enzyme- mediated nucleic acid synthesis. The following are non-limiting embodiments of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A nucleic acid molecule may also comprise modified nucleic acid molecules, such as methylated nucleic acid molecules and nucleic acid molecule analogs. Analogs of purines and pyrimidines are known in the art, and include, but are not limited to, aziridinycytosine, 4-acetylcytosine, 5-ffuorouracil, 5-bromouracil, 5-carboxymethylamino- methyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, inosine, N6-isopentenyladenine, 1-methyl- adenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyl- adenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, pseudouracil, 5-pentylnyluracil and 2,6-diaminopurine. The use of uracil as a substitute for thymine in a deoxyribonucleic acid is also considered an analogous form of pyrimidine. A "fragment" of a polynucleotide is a polynucleotide comprised of at least 9 contiguous nucleotides, preferably at least 15 contiguous nucleotides and more preferably at least 45 nucleotides, of coding or non-coding sequences.

The term "gene targeting" refers to a type of homologous recombination that occurs when a fragment of genomic DNA is introduced into a mammalian cell and that fragment locates and recombines with endogenous homologous sequences.

The term "homologous recombination" refers to the exchange of DNA fragments between two DNA molecules or chromatids at the site of homologous nucleotide sequences. The term "homologous" as used herein denotes a characteristic of a DNA sequence having at least about 70 percent sequence identity as compared to a reference sequence, typically at least about 85 percent sequence identity, preferably at least about 95 percent sequence identity, and more preferably about 98 percent sequence identity, and most preferably about 100 percent sequence identity as compared to a reference sequence. Homology can be determined using a "BLASTN" algorithm. It is understood that homologous sequences can accommodate insertions, deletions and substitutions in the nucleotide sequence. Thus, linear sequences of nucleotides can be essentially identical even if some of the nucleotide residues do not precisely correspond or align. The reference sequence may be a subset of a larger sequence, such as a portion of a gene or flanking sequence, or a repetitive portion of a chromosome.

As used herein, the term "target gene" (alternatively referred to as "target gene sequence" or "target DNA sequence" or "target sequence") or "target GPCR gene" refers to any nucleic acid molecule or polynucleotide of any gene to be modified by homologous recombination. The target sequence includes an intact gene, an exon or intron, a regulatory sequence or any region between genes. The target gene comprises a portion of a particular gene or genetic locus in the individual's genomic DNA. As provided herein, the target gene of the present invention, in alternate embodiments, is a GPCR gene selected from the following: DEZ (or CMKLRl) orphan receptor;

An "adrenomedullin receptor gene" refers to a nucleotide sequence that encodes an adrenomedullin receptor, such as a sequence comprising SEQ ID NO:7, identified in GenBank as Accession number: AF001229; GI/MD number: 2150047.

A "CRFR2 gene" refers to a nucleotide sequence that encodes a CRFR2 corticotropin- releasing factor receptor, such as a sequence comprising SEQ ID NO: 10, identified in GenBank as Accession number: U21729; GI/MD number: 717137.

A "FPRL3 gene" refers to a nucleotide sequence that encodes a FPRL3 N-formylpeptide receptor, such as a sequence comprising SEQ ID NO: 13, identified in GenBank as Accession number: AF071181; GI/MD number: 3549281. A "NPY4-R gene" refers to a nucleotide sequence that encodes a NPY4-R neuropeptide Y receptor, such as a sequence comprising SEQ ID NO: 16, identified in GenBank as Accession number: U40189; GI/MD number: 1223969.

A "NPY6-R gene" refers to a nucleotide sequence that encodes a NPY6-R neuropeptide Y receptor, such as a sequence comprising SEQ ID NO: 19, identified in GenBank as Accession number: U58367; GI/NID number: 1378003.

A "KOR3 gene" refers to a nucleotide sequence that encodes a KOR3 opiate receptor, such as a sequence comprising SEQ ID NO:22, identified in GenBank as Accession number: U09421; GI/MD number: 551484.

An "mGluR8 gene" refers to a nucleotide sequence that encodes an mGluR8 (or MGR8) metabotropic glutamate receptor, such as a sequence comprising SEQ ID NO:25, identified in GenBank as Accession number: U17252; GI/NID number: 854728.

A "MAS receptor gene" refers to a nucleotide sequence that encodes a MAS receptor, such as a sequence comprising SEQ ID NO:28, identified in GenBank as Accession number: X67735; GI/NID number: 53011. "Disruption" of a target gene occurs when a fragment of genomic DNA locates and recombines with an endogenous homologous sequence. These sequence disruptions or modifications may include insertions, missense, frameshift, deletion, or substitutions, or replacements of DNA sequence, or any combination thereof. Insertions include the insertion of entire genes which may be of animal, plant, prokaryotic, or viral origin. Disruption, for example, can alter or replace a promoter, enhancer, or splice site of a target gene, and can alter the normal gene product by inhibiting its production partially or completely or by enhancing the normal gene product's activity.

The term, "transgenic cell", refers to a cell containing within its genome a target gene that has been disrupted, modified, altered, or replaced completely or partially by the method of gene targeting. The term "transgenic animal" refers to an animal that contains within its genome a specific gene that has been disrupted by the method of gene targeting. The transgenic animal includes both the heterozygote animal (i.e., one defective allele and one wild-type allele) and the homozygous animal (i.e., two defective alleles).

As used herein, the terms "selectable marker" or "positive selection marker" refers to a gene encoding a product that enables only the cells that carry the gene to survive and/or grow under certain conditions. For example, plant and animal cells that express the introduced neomycin resistance

(Neo¹) gene are resistant to the compound G418. Cells that do not carry the Neo^r gene marker are killed by G418. Other positive selection markers will be known to those of skill in the art.

A "host cell" includes an individual cell or cell culture which can be or has been a recipient for vector(s) or for incorporation of nucleic acid molecules and or proteins. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent due to natural, accidental, or deliberate mutation. A host cell includes cells transfected with the constructs of the present invention. The term "modulates" as used herein refers to the inhibition, reduction, increase or enhancement of a target gene or target protein function, expression, activity, or alternatively a phenotype associated with a disruption in a target gene. In a preferred embodiment, the term "modulates" refers to agents that act as agonists or antagonists of the target GPCR.

The term "ameliorates" refers to a decreasing, reducing or eliminating of a condition, disease, disorder, or phenotype, including an abnormality or symptom associated with a disruption in a target gene. The term "abnormality" refers to any disease, disorder, condition, or phenotype in which a disruption of a target gene is implicated, including pathological conditions.

Brief Description of the Drawings Figure 1 shows a polynucleotide sequence for a DEZ orphan receptor gene (SEQ ID NO: 1). Figures 2 and 3 show the design of the targeting construct used to disrupt a DEZ orphan receptor gene, as well as the location and extent of the deletion in the target sequence. Figure 3 shows the sequences identified as SEQ ID NO:2 and SEQ ID NO:3, which were used as the targeting arms (homologous sequences) in the DEZ orphan receptor gene targeting construct.

Figure 4 shows the polynucleotide sequence for a CB2R gene (SEQ ID NO:4). Figures 5 and 6 show the design of the targeting construct used to disrupt a CB2R gene, as well as the location and extent of the deletion in the target sequence. Figure 6 shows the sequences identified as SEQ ID NO:5 and SEQ ID NO:6, which were used as the targeting arms (homologous sequences) in the CB2R gene targeting construct.

Figure 7 shows the polynucleotide sequence for an adrenomedullin receptor gene (SEQ ID NO:7). Figures 8 and 9 show the design of the targeting construct used to disrupt an adrenomedullin receptor gene, as well as the location and extent of the deletion in the target sequence. Figure 9 shows the sequences identified as SEQ ID N0:8 and SEQ ID N0:9, which were used as the targeting arms (homologous sequences) in the adrenomedullin receptor gene targeting construct

Figure 10 shows the polynucleotide sequence for a CRFR2 gene (SEQ ID NO: 10). Figures 11 and 12 show the design of the targeting construct used to disrupt a CRFR2 gene, as well as the location and extent of the deletion in the target sequence. Figure 12 shows the sequences identified as SEQ ID NO: 11 and SEQ ID NO: 12, which were used as the targeting arms (homologous sequences) in the CRFR2 gene targeting construct.

Figure 13 shows the polynucleotide sequence of a FPRL3 gene (SEQ ID NO: 13). Figures 14 and 15 show the design of the targeting construct used to disrupt an FPRL3 gene, as well as the location and extent of the deletion in the target sequence. Figure 15 shows the sequences identified as SEQ ID NO: 14 and SEQ ID NO: 15 which were used as the targeting arms (homologous sequences) in the FPRL3 gene targeting construct.

Figure 16 shows the polynucleotide sequence for a NPY4-R gene (SEQ ID NO: 16). Figures 17 and 18 show the design of the targeting construct used to disrupt an NPY4-R gene, as well as the location and extent of the deletion in the target sequence. Figure 18 shows the sequences identified as SEQ ID NO: 17 and SEQ ID NO: 18, which were used as the targeting arms (homologous sequences) in the NPY4-R gene targeting construct.

Figure 19 shows the polynucleotide sequence for a NPY6-R gene (SEQ ID NO: 19). Figures 20 and 21 show the design of the targeting construct used to disrupt an NPY6-R gene, as well as the location and extent of the deletion in the target sequence. Figure 21 shows the sequences identified as SEQ ID NO: 20 and SEQ ID NO:21, which were used as the targeting arms (homologous sequences) in the NPY6-R gene targeting construct.

Figure 22 shows the polynucleotide sequence for a KOR3 gene (SEQ ID NO:22). Figures 23 and 24 show the design of the targeting construct used to disrupt a KOR3 gene, as well as the location and extent of the deletion in the target sequence. Figure 24 shows the sequences identified as SEQ ID NO: 23 and SEQ ID NO: 24, which were used as the targeting arms (homologous sequences) in the KOR3 gene targeting construct.

Figure 25 shows the polynucleotide sequence for a mGluR8 gene (SEQ ID NO:25). Figures 26 and 27 show the design of the targeting construct used to disrupt an mGluR8 gene, as well as the location and extent of the deletion in the target sequence. Figure 27 shows the sequences identified as SEQ ID NO:26 and SEQ ID NO:27, which were used as the targeting arms (homologous sequences) in the mGluR8 gene targeting construct.

Figure 28 shows the polynucleotide sequence for a MAS receptor gene (SEQ ID NO:28). Figures 29 and 30 show the design of the targeting construct used to disrupt an MAS receptor gene, as well as the location and extent of the deletion in the target sequence. Figure 30 shows the sequences identified as SEQ ID NO:29 and SEQ ID NO:30, which were used as the targeting arms (homologous sequences) in the MAS receptor gene targeting construct.

Detailed Description of the Invention

The invention is based, in part, on the evaluation of the expression and role of genes and gene expression products, primarily those associated with a target gene. Among others, the invention permits the definition of disease pathways and the identification of diagnostically and therapeutically useful targets. For example, genes which are mutated or down-regulated under disease conditions may be involved in causing or exacerbating the disease condition. Treatments directed at up- regulating the activity of such genes or treatments which involve alternate pathways, may ameliorate the disease condition.

Generation of Targeting Construct

The targeting construct of the present invention may be produced using standard methods known in the art. (See, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; E.N. Glover (eds.), 1985, DNA Cloning: A Practical Approach, Volumes I and II; M.J. Gait (ed.), 1984,

Oligonucleotide Synthesis; B.D. Hames & SJ. Higgins (eds.), 1985, Nucleic Acid Hybridization; B.D.

Hames & SJ. Higgins (eds.), 1984, Transcription and Translation; R.I. Freshney (ed.), 1986, Animal

Cell Culture; Immobilized Cells and Enzymes, IRL Press, 1986; B. Perbal, 1984, A Practical Guide To Molecular Cloning; F.M. Ausubel et al, 1994, Current Protocols in Molecular Biology, lohn Wiley & Sons, Inc.). For example, the targeting construct may be prepared in accordance with conventional ways, where sequences may be synthesized, isolated from natural sources, manipulated, cloned, ligated, subjected to in vitro mutagenesis, primer repair, or the like. At various stages, the joined sequences may be cloned, and analyzed by restriction analysis, sequencing, or the like.

The targeting DNA can be constructed using techniques well known in the art. For example, the targeting DNA may be produced by chemical synthesis of oligonucleotides, nick-translation of a double-stranded DNA template, polymerase chain-reaction amplification of a sequence (or ligase chain reaction amplification), purification of prokaryotic or target cloning vectors harboring a sequence of interest (e.g., a cloned cDNA or genomic DNA, synthetic DNA or from any of the aforementioned combination) such as plasmids, phagemids, YACs, cosmids, bacteriophage DNA, other viral DNA or replication intermediates, or purified restriction fragments thereof, as well as other sources of single and double-stranded polynucleotides having a desired nucleotide sequence. Moreover, the length of homology may be selected using known methods in the art. For example, selection may be based on the sequence composition and complexity of the predetermined endogenous target DNA sequence(s). The targeting construct of the present invention typically comprises a first sequence homologous to a portion or region of the target gene and a second sequence homologous to a second portion or region of the target gene. The targeting construct further comprises a positive selection marker, which is preferably positioned in between the first and the second DNA sequence that are homologous to a portion or region of the target DNA sequence. The positive selection marker may be operatively linked to a promoter and a polyadenylation signal. Other regulatory sequences known in the art may be incorporated into the targeting construct to disrupt or control expression of a particular gene in a specific cell type. In addition, the targeting construct may also include a sequence coding for a screening marker, for example, green fluorescent protein (GFP), or another modified fluorescent protein.

Although the size of the homologous sequence is not critical and can range from as few as 50 base pairs to as many as 100 kb, preferably each fragment is greater than about 1 kb in length, more preferably between about 1 and about 10 kb, and even more preferably between about 1 and about 5 kb. One of skill in the art will recognize that although larger fragments may increase the number of homologous recombination events in ES cells, larger fragments will also be more difficult to clone.

In a preferred embodiment of the present invention, the targeting construct is prepared directly from a plasmid genomic library using the methods described in pending U.S. Patent Application No.: 08/971,310, filed November 17, 1997, the disclosure of which is incorporated herein in its entirety. Generally, a sequence of interest is identified and isolated from a plasmid library in a single step using, for example, long-range PCR. Following isolation of this sequence, a second polynucleotide that will disrupt the target sequence can be readily inserted between two regions encoding the sequence of interest. In accordance with this aspect, the construct is generated in two steps by (1) amplifying (for example, using long-range PCR) sequences homologous to the target sequence, and (2) inserting another polynucleotide (for example a selectable marker) into the PCR product so that it is flanked by the homologous sequences. Typically, the vector is a plasmid from a plasmid genomic library. The completed construct is also typically a circular plasmid. In another embodiment, the targeting construct is designed in accordance with the regulated positive selection method described in U.S. Application No. 60/232,957, filed September 15, 2000, the disclosure of which is incorporated herein in its entirety. The targeting construct is designed to include a PGK-/zeo fusion gene having two lacO sites, positioned in the PGK promoter and an NLS- lacl gene comprising a lac repressor fused to sequences encoding the NLS from the SV40 T antigen. In another embodiment, the targeting construct may contain more than one selectable maker gene, including a negative selectable marker, such as the herpes simplex virus tk (HSV-tk) gene. The negative selectable marker may be operatively linked to a promoter and a polyadenylation signal. (See, e.g., U.S. Patent No. 5,464,764; U.S. Patent No. 5,487,992; U.S. Patent No. 5,627,059; and U.S. Patent No. 5,631,153). 49.

Generation of Cells and Confirmation of Homologous Recombination Events

Once an appropriate targeting construct has been prepared, the targeting construct may be introduced into an appropriate host cell using any method known in the art. Various techniques may be employed in the present invention, including, for example, pronuclear microinjection; retro virus mediated gene transfer into germ lines; gene targeting in embryonic stem cells; electroporation of embryos; sperm-mediated gene transfer; and calcium phosphate/DNA co-precipitates, microinjection of DNA into the nucleus, bacterial protoplast fusion with intact cells, transfection, polycations, e.g., polybrene, polyornithine, etc., or the like (See, e.g., U.S. Pat. No. 4,873,191; Van der Putten, et al.,

1985, Proc. Natl. Acad. Sci, USA 82:6148-6152; Thompson, et al, 1989, Cell 56:313-321; Lo, 1983, Mol Cell. Biol. 3: 1803-1814; Lavitrano, et al.,1989, Cell, 57:717-723). Various techniques for transforming mammalian cells are known in the art. (See, e.g., Gordon, 1989, Intl. Rev. Cytol, 115:171-229; Keown et al., 1989, Methods in Enzymology; Keown et al, 1990, Methods and Enzymology, Vol. 185, pp. 527-537; Mansour et al, 1988, Nature, 336:348-352).

In a preferred aspect of the present invention, the targeting construct is introduced into host cells by electroporation. In this process, electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the construct. The pores created during electroporation permit the uptake of macromolecules such as DNA. (See, e.g., Potter, H., et al., 1984, Proc. Natl Acad. Sci. U.S.A. 81:7161-7165).

Any cell type capable of homologous recombination may be used in the practice of the present invention. Examples of such target cells include cells derived from vertebrates including mammals such as humans, bovine species, ovine species, murine species, simian species, and ether eucaryotic organisms such as filamentous fungi, and higher multicellular organisms such as plants.

Preferred cell types include embryonic stem (ES) cells, which are typically obtained from pre- implantation embryos cultured in vitro. (See, e.g., Evans, M. J., et al., 1981, Nature 292: 154-156; Bradley, M. O., et al, 1984, Nature 309:255-258; Gossler et al, 1986, Proc. Natl. Acad. Sci. USA 83:9065-9069; and Robertson, et al., 1986, Nature 322:445-448). The ES cells are cultured and prepared for introduction of the targeting construct using methods well known to the skilled artisan. (See, e.g., Robertson, E. J. ed. "Teratocarcinomas and Embryonic Stem Cells, a Practical Approach", IRL Press, Washington D.C., 1987; Bradley et al, 1986, Current Topics in Devel. Biol. 20:357-371; by Hogan et al. in "Manipulating the Mouse Embryo": A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y., 1986; Thomas et al., 1987, Cell 51:503; Koller et al, 1991, Proc. Natl. Acad. Sci. USA, 88:10730; Dorin et al, 1992, Transgenic Res. 1:101; and Veis et al, 1993, Cell 75:229). The ES cells that will be inserted with the targeting construct are derived from an embryo or blastocyst of the same species as the developing embryo into which they are to be introduced. ES cells are typically selected for their ability to integrate into the inner cell mass and contribute to the germ line of an individual when introduced into the mammal in an embryo at the blastocyst stage of development. Thus, any ES cell line having this capability is suitable for use in the practice of the present invention.

The present invention may also be used to knockout genes in other cell types, such as stem cells. By way of example, stem cells may be myeloid, lymphoid, or neural progenitor and precursor cells. These cells comprising a disruption or knockout of a gene may be particularly useful in the study of target gene function in individual developmental pathways. Stem cells may be derived from any vertebrate species, such as mouse, rat, dog, cat, pig, rabbit, human, non-human primates and the like.

After the targeting construct has been introduced into cells, the cells where successful gene targeting has occurred are identified. Insertion of the targeting construct into the targeted gene is typically detected by identifying cells for expression of the marker gene. In a preferred embodiment, the cells transformed with the targeting construct of the present invention are subjected to treatment with an appropriate agent that selects against cells not expressing the selectable marker. Only those cells expressing the selectable marker gene survive and/or grow under certain conditions. For example, cells that express the introduced neomycin resistance gene are resistant to the compound G418, while cells that do not express the neo gene marker are killed by G418. If the targeting construct also comprises a screening marker such as GFP, homologous recombination can be identified through screening cell colonies under a fluorescent light. Cells that have undergone homologous recombination will have deleted the GFP gene and will not fluoresce. If a regulated positive selection method is used in identifying homologous recombination events, the targeting construct is designed so that the expression of the selectable marker gene is regulated in a manner such that expression is inhibited following random integration but is permitted (derepressed) following homologous recombination. More particularly, the transfected cells are screened for expression of the neo gene, which requires that (1) the cell was successfully electroporated, and (2) lac repressor inhibition of neo transcription was relieved by homologous recombination. This method allows for the identification of transfected cells and homologous recombinants to occur in one step with the addition of a single drug.

Alternatively, a positive-negative selection technique may be used to select homologous recombinants. This technique involves a process in which a first drug is added to the cell population, for example, a neomycin-like drug to select for growth of transfected cells, i.e. positive selection. A second drug, such as FIAU is subsequently added to kill cells that express the negative selection marker, i.e. negative selection. Cells that contain and express the negative selection marker are killed by a selecting agent, whereas cells that do not contain and express the negative selection marker survive. For example, cells with non-homologous insertion of the construct express HSV thymidine kinase and therefore are sensitive to the herpes drugs such as gancyclovir (GANC) or FIAU (l-(2- deoxy 2-fluoro-B-D-arabinofluranosyl)-5-iodouracil). (See, e.g., Mansour et al., Nature 336:348-352: (1988); Capecchi, Science 244: 1288-1292, (1989); Capecchi, Trends in Genet. 5:70-76 (1989)).

Successful recombination may be identified by analyzing the DNA of the selected cells to confirm homologous recombination. Various techniques known in the art, such as PCR and or Southern analysis may be used to confirm homologous recombination events.

Homologous recombination may also be used to disrupt genes in stem cells, and other cell types, which are not totipotent embryonic stem cells. By way of example, stem cells may be myeloid, lymphoid, or neural progenitor and precursor cells. Such transgenic cells may be particularly useful in the study of gene function in individual developmental pathways. Stem cells may be derived from any vertebrate species, such as mouse, rat, dog, cat, pig, rabbit, human, non-human primates and the like.

In cells which are not totipotent it may be desirable to knock out both copies of the target using methods which are known in the art. For example, cells comprising homologous recombination at a target locus which have been selected for expression of a positive selection marker (e.g., Neo¹) and screened for non-random integration, can be further selected for multiple copies of the selectable marker gene by exposure to elevated levels of the selective agent (e.g., G418). The cells are then analyzed for homozygosity at the target locus. Alternatively, a second construct can be generated with a different positive selection marker inserted between the two homologous sequences. The two constructs can be introduced into the cell either sequentially or simultaneously, followed by appropriate selection for each of the positive marker genes. The final cell is screened for homologous recombination of both alleles of the target. Production of Transgenic Animals

Selected cells are then injected into a blastocyst (or other stage of development suitable for the purposes of creating a viable animal, such as, for example, a morula) of an animal (e.g., a mouse) to form chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed., IRL, Oxford, pp. 113-152 (1987)). Alternatively, selected ES cells can be allowed to aggregate with dissociated mouse embryo cells to form the aggregation chimera. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Chimeric progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA. In one embodiment, chimeric progeny mice are used to generate a mouse with a heterozygous disruption in the gene. Heterozygous transgenic mice can then be mated. It is well know in the art tiiat typically VΛ of the offspring of such matings will have a homozygous disruption in the target gene. The heterozygous and homozygous transgenic mice can then be compared to normal, wild type mice to determine whether disruption of the target gene causes phenotypic changes, especially pathological changes. For example, heterozygous and homozygous mice may be evaluated for phenotypic changes by physical examination, necropsy, histology, clinical chemistry, complete blood count, body weight, organ weights, and cytological evaluation of bone marrow.

In one embodiment, the phenotype (or phenotypic change) associated with a disruption in the target gene is placed into or stored in a database. Preferably, the database includes: (i) genotypic data

(e.g., identification of the disrupted gene) and (ii) phenotypic data (e.g., phenotype(s) resulting from the gene disruption) associated with the genotypic data. The database is preferably electronic. In addition, the database is preferably combined with a search tool so that the database is searchable.

Conditional Trans enic Animals The present invention further contemplates conditional transgenic or knockout animals, such as those produced using recombination methods. Bacteriophage PI Cre recombinase and flp recombinase from yeast plasmids are two non-limiting examples of site-specific DNA recombinase enzymes which cleave DNA at specific target sites (lox P sites for cre recombinase and fit sites for flp recombinase) and catalyze a ligation of this DNA to a second cleaved site. A large number of suitable alternative site-specific recombinases have been described, and their genes can be used in accordance with the method of the present invention. Such recombinases include the Int recombinase of bacteriophage λ (with or without Xis) (Weisberg, R. et. al., in Lambda II, (Hendrix, R., et al, Eds.), Cold Spring Harbor Press, Cold Spring Harbor, NY, pp. 211-50 (1983), herein incorporated by reference); Tpnl and the β-lactamase transposons (Mercier, et al., J. Bacteriol, 172:3745-57 (1990)); the Tn3 resolvase (Flanagan & Fennewald J. Molec. Biol, 206:295-304 (1989); Stark, et al, Cell, 58:779-90 (1989)); the yeast recombinases (Matsuzaki, et al, J. Bacteriol, 172:610-18 (1990)); the B. subtilis SpoIVC recombinase (Sato, et al, J. Bacteriol. 172:1092-98 (1990)); the Flp recombinase (Schwartz & Sadowski, /. Molec.BioL, 205:647-658 (1989); Parsons, et al, J. Biol. Chem., 265:4527-33 (1990); Golic & Lindquist, Cell, 59:499-509 (1989); Amin, et al, J. Molec. Biol, 214:55-72 (1990)); the Hin recombinase (Glasgow, et al, J. Biol. Chem., 264:10072-82 (1989)); immunoglobulin recombinases (Malynn, et al, Cell, 54:453-460 (1988)); and the Cin recombinase (Haffter & Bickle, EMBO J., 7:3991-3996 (1988); Hubner, et al, J. Molec. Biol, 205:493-500 (1989)), all herein incorporated by reference. Such systems are discussed by Echols (/. Biol. Chem. 265: 14697-14700 (1990)); de Villartay (Nature, 335:170-74 (1988)); Craig, (Ann. Rev. Genet., 22:77-105 (1988)); Poyart-Salmeron, et al, (EMBO J. 8:2425-33 (1989)); Hunger-Bertling, et al. (Mol Cell. Biochem., 92:107-16 (1990)); and Cregg & Madden (Mol. Gen. Genet, 219:320-23 (1989)), all herein incorporated by reference.

Cre has been purified to homogeneity, and its reaction with the loxP site has been extensively characterized (Abremski & Hess /. Mol. Biol. 259:1509-14 (1984), herein incorporated by reference). Cre protein has a molecular weight of 35,000 and can be obtained commercially from New England Nuclear/DuPont. The cre gene (which encodes the Cre protein) has been cloned and expressed (Abremski, et al Cell 32:1301-11 (1983), herein incorporated by reference). The Cre protein mediates recombination between two loxP sequences (Sternberg, et al. Cold Spring Harbor Symp. Quant. Biol. 45:297-309 (1981)), which may be present on the same or different DNA molecule. Because the internal spacer sequence of the loxP site is asymmetrical, two loxP sites can exhibit directionality relative to one another (Hoess & Abremski Proc. Natl. Acad. Sci. U.S.A. 81:1026-29 (1984)). Thus, when two sites on the same DNA molecule are in a directly repeated orientation, Cre will excise the DNA between the sites (Abremski, et al. Cell 32:1301-11 (1983)). However, if the sites are inverted with respect to each other, the DNA between them is not excised after recombination but is simply inverted. Thus, a circular DNA molecule having two loxP sites in direct orientation will recombine to produce two smaller circles, whereas circular molecules having two loxP sites in an inverted orientation simply invert the DNA sequences flanked by the loxP sites. In addition, recombinase action can result in reciprocal exchange of regions distal to the target site when targets are present on separate DNA molecules.

Recombinases have important application for characterizing gene function in knockout models. When the constructs described herein are used to disrupt target genes, a fusion transcript can be produced when insertion of the positive selection marker occurs downstream (3') of the translation initiation site of the target gene. The fusion transcript could result in some level of protein expression with unknown consequence. It has been suggested that insertion of a positive selection marker gene can affect the expression of nearby genes. These effects may make it difficult to determine gene function after a knockout event since one could not discern whether a given phenotype is associated with the inactivation of a gene, or the transcription of nearby genes. Both potential problems are solved by exploiting recombinase activity. When the positive selection marker is flanked by recombinase sites in the same orientation, the addition of the corresponding recombinase will result in the removal of the positive selection marker. In this way, effects caused by the positive selection marker or expression of fusion transcripts are avoided. In one embodiment, purified recombinase enzyme is provided to the cell by direct microinjection. In another embodiment, recombinase is expressed from a co-transfected construct or vector in which the recombinase gene is operably linked to a functional promoter. An additional aspect of this embodiment is the use of tissue-specific or inducible recombinase constructs which allow the choice of when and where recombination occurs. One method for practicing the inducible forms of recombinase-mediated recombination involves the use of vectors that use inducible or tissue- specific promoters or other gene regulatory elements to express the desired recombinase activity. The inducible expression elements are preferably operatively positioned to allow the inducible control or activation of expression of the desired recombinase activity. Examples of such inducible promoters or other gene regulatory elements include, but are not limited to, tetracycline, metallothionine, ecdysone, and other steroid-responsive promoters, rapamycin responsive promoters, and the like (No, et al. Proc. Natl. Acad. Sci. USA, 93:3346-51 (1996); Furth, et al. Proc. Natl. Acad. Sci. USA, 91:9302-6 (1994)). Additional control elements that can be used include promoters requiring specific transcription factors such as viral, promoters. Vectors incorporating such promoters would only express recombinase activity in cells that express the necessary transcription factors. Models for Disease The cell- and animal-based systems described herein can be utilized as models for diseases.

Animals of any species, including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate disease animal models. In addition, cells from humans may be used. These systems may be used in a variety of applications. Such assays may be utilized as part of screening strategies designed to identify agents, such as compounds which are capable of ameliorating disease symptoms. Thus, the animal- and cell-based models may be used to identify drugs, pharmaceuticals, therapies and interventions which may be effective in treating disease.

Cell-based systems may be used to identify compounds which may act to ameliorate disease symptoms. For example, such cell systems may be exposed to a compound suspected of exhibiting an ability to ameliorate disease symptoms, at a sufficient concentration and for a time sufficient to elicit such an amelioration of disease symptoms in the exposed cells. After exposure, the cells are examined to determine whether one or more of the disease cellular phenotypes has been altered to resemble a more normal or more wild type, non-disease phenotype.

In addition, animal-based disease systems, such as those described herein, may be used to identify compounds capable of ameliorating disease symptoms. Such animal models may be used as test substrates for the identification of drugs, pharmaceuticals, therapies, and interventions which may be effective in treating a disease or other phenotypic characteristic of the animal. For example, animal models may be exposed to a compound or agent suspected of exhibiting an ability to ameliorate disease symptoms, at a sufficient concentration and for a time sufficient to elicit such an amelioration of disease symptoms in the exposed animals. The response of the animals to the exposure may be monitored by assessing the reversal of disorders associated with the disease. Exposure may involve treating mother animals during gestation of the model animals described herein, thereby exposing embryos or fetuses to the compound or agent which may prevent or ameliorate the disease or phenotype. Neonatal, juvenile, and adult animals can also be exposed. More particularly, using the animal models of the invention, specifically, transgenic mice, methods of identifying agents, including compounds are provided, preferably, on the basis of the ability to affect at least one phenotype associated with a disruption in a target gene. In one embodiment, the present invention provides a method of identifying agents having an effect on target gene or alternatively, target protein expression or function. The method includes measuring a physiological response of the animal, for example, to the agent, and comparing the physiological response of such animal to a control animal, wherein the physiological response of the animal comprising a disruption in a target gene as compared to the control animal indicates the specificity of the agent. A "physiological response" is any biological or physical parameter of an animal which can be measured. Molecular assays (e.g., gene transcription, protein production and degradation rates), physical parameters (e.g., exercise physiology tests, measurement of various parameters of respiration, measurement of heart rate or blood pressure, measurement of bleeding time, aPTT.T, or TT), and cellular assays (e.g.,. immunohistochemical assays of cell surface markers, or the ability of cells to aggregate or proliferate) can be used to assess a physiological response. The transgenic animals and cells of the present invention may by utilized as models for diseases, disorders, or conditions associated with phenotypes relating to a disruption in a target gene. The present invention provides a unique animal model for testing and developing new treatments relating to the behavioral phenotypes. Analysis of the behavioral phenotype allows for the development of an animal model useful for testing, for instance, the efficacy of proposed genetic and pharmacological therapies for human genetic diseases, such as neurological, neuropsychological, or psychotic illnesses. A statistical analysis of the various behaviors measured can be carried out using any conventional statistical program routinely used by those skilled in the art (such as, for example, "Analysis of Variance" or ANOVA). A "p" value of about 0.05 or less is generally considered to be statistically significant, although slightly higher p values may still be indicative of statistically significant differences. To statistically analyze abnormal behavior, a comparison is made between the behavior of a transgenic animal (or a group thereof) to the behavior of a wild-type mouse (or a group thereof), typically under certain prescribed conditions. "Abnormal behavior" as used herein refers to behavior exhibited by an animal having a disruption in the target gene, e.g. transgenic animal, which differs from an animal without a disruption in the target gene, e.g. wild-type mouse. Abnormal behavior consists of any number of standard behaviors that can be objectively measured (or observed) and compared. In the case of comparison, it is preferred that the change be statistically significant to confirm that there is indeed a meaningful behavioral difference between the knockout animal and the wild-type control animal. Examples of behaviors which may be measured or observed include, but are not limited to, ataxia, rapid limb movement, eye movement, breathing, motor activity, cognition, emotional behaviors, social behaviors, hyperactivity, hypersensitivity, anxiety, impaired learning, abnormal reward behavior, and abnormal social interaction, such as aggression.

A series of tests may be used to measure the behavioral phenotype of the animal models of the present invention, including neurological and neuropsychological tests to identify abnormal behavior. These tests may be used to measure abnormal behavior relating to, for example, learning and memory, eating, pain, aggression, sexual reproduction, anxiety, depression, schizophrenia, and drug abuse. (See, e.g., Crawley and Paylor, Hormones and Behavior 31:197-211 (1997)). The social interaction test involves exposing a mouse to other animals in a variety of settings.

The social behaviors of the animals (e.g., touching, climbing, sniffing, and mating) are subsequently evaluated. Differences in behaviors can then be statistically analyzed and compared (See, e.g., S. E.

File, et al, Pharmacol. Bioch. Behav. 22:941-944 (1985); R. R. Holson, Phys. Behav. 37:239-247 (1986)). Examplary behavioral tests include the following.

The mouse startle response test typically involves exposing the animal to a sensory (typically auditory) stimulus and measuring the startle response of the animal (see, e.g., M. A. Geyer, et al, Brain Res. Bull. 25:485-498 (1990); Paylor and Crawley, Psychopharmacology 132:169-180 (1997)). A pre-pulse inhibition test can also be used, in which the percent inhibition (from a normal startle response) is measured by "cueing" the animal first with a brief low-intensity pre-pulse prior to the startle pulse.

The electric shock test generally involves exposure to an electrified surface and measurement of subsequent behaviors such as, for example, motor activity, learning, social behaviors. The behaviors are measured and statistically analyzed using standard statistical tests. (See, e.g., G. J. Kant, et al, Pharm. Bioch. Behav. 20:793-797 (1984); N. J. Leidenheimer, et al, Pharmacol. Bioch. Behav. 30:351-355 (1988)).

The tail-pinch or immobilization test involves applying pressure to the tail of the animal and/or restraining the animal's movements. Motor activity, social behavior, and cognitive behavior are examples of the areas that are measured. (See, e.g., M. Bertolucci D'Angic, et al, Neurochem. 55:1208-1214 (1990)).

The novelty test generally comprises exposure to a novel environment and/or novel objects.

The animal's motor behavior in the novel environment and/or around the novel object are measured and statistically analyzed. (See, e.g., D. K. Reinstein, et al, Pharm. Bioch. Behav. 17:193-202 (1982);

B. Poucet, Behav. Neurosci. 103:1009-10016 (1989); R. R. Holson, et al, Phys. Behav. 37:231-238 (1986)). This test may be used to detect visual processing deficiencies or defects.

The learned helplessness test involves exposure to stresses, for example, noxious stimuli, which cannot be affected by the animal's behavior. The animal's behavior can be statistically analyzed using various standard statistical tests. (See, e.g., A. Leshner, et al, Behav. Neural Biol. 26:497-501 (1979)). Alternatively, a tail suspension test may be used, in which the "immobile" time of the mouse is measured when suspended "upside-down" by its tail. This is a measure of whether the animal struggles, an indicator of depression. In humans, depression is believed to result from feelings of a lack of control over one's life or situation. It is believed that a depressive state can be elicited in animals by repeatedly subjecting them to aversive situations over which they have no control. A condition of "learned helplessness" is eventually reached, in which the animal will stop trying to change its circumstances and simply accept its fate. Animals that stop struggling sooner are believed to be more prone to depression. Studies have shown that the administration of certain antidepressant drugs prior to testing increases the amount of time that animals struggle before giving up.

The Morris water-maze test comprises learning spatial orientations in water and subsequently measuring the animal's behaviors, such as, for example, by counting the number of incorrect choices. The behaviors measured are statistically analyzed using standard statistical tests. (See, e.g., E. M. Spruijt, et al, Brain Res. 527:192-197 (1990)).

Alternatively, a Y-shaped maze may be used (see, e.g., McFarland, D.J., Pharmacology, Biochemistry and Behavior 32:723-726 (1989); Delhi, F., et al., Neurobiology of Learning and Memory 73:31-48 (2000)). The Y-maze is generally believed to be a test of cognitive ability. The dimensions of each arm of the Y-maze can be, for example, approximately 40 cm x 8 cm x 20 cm, although other dimensions may be used. Each arm can also have, for example, sixteen equally spaced photobeams to automatically detect movement within the arms. At least two different tests can be performed using such a Y-maze. In a continuous Y-maze paradigm, mice are allowed to explore all three arms of a Y-maze for, e.g., approximately 10 minutes. The animals are continuously tracked using photobeam detection grids, and the data can be used to measure spontaneous alteration and positive bias behavior. Spontaneous alteration refers to the natural tendency of a "normal" animal to visit the least familiar arm of a maze. An alternation is scored when the animal makes two consecutive turns in the same direction, thus representing a sequence of visits to the least recently entered arm of the maze. Position bias determines egocentrically defined responses by measuring the animal's tendency to favor turning in one direction over another. Therefore, the test can detect differences in an animal's ability to navigate on the basis of allocentric or egocentric mechanisms. The two-trial Y-maze memory test measures response to novelty and spatial memory based on a free- choice exploration paradigm. During the first trial (acquisition), the animals are allowed to freely visit two arms of the Y-maze for, e.g., approximately 15 minutes. The third arm is blocked off during this trial. The second trial (retrieval) is performed after an intertrial interval of, e.g., approximately 2 hours. During the retrieval trial, the blocked arm is opened and the animal is allowed access to all three arms for, e.g., approximately 5 minutes. Data are collected during the retrieval trial and analyzed for the number and duration of visits to each arm. Because the three arms of the maze are virtually identical, discrimination between novelty and familiarity is dependent on "environmental" spatial cues around the room relative to the position of each arm. Changes in arm entry and duration of time spent in the novel arm in a transgenic animal model may be indicative of a role of that gene in mediating novelty and recognition processes.

The passive avoidance or shuttle box test generally involves exposure to two or more environments, one of which is noxious, providing a choice to be learned by the animal. Behavioral measures include, for example, response latency, number of correct responses, and consistency of response. (See, e.g., R. Ader, et al, Psychon. Sci. 26:125-128 (1972); R. R. Holson, Phys. Behav. 37:221-230 (1986)). Alternatively, a zero-maze can be used. In a zero-maze, the animals can, for example, be placed in a closed quadrant of an elevated annular platform having, e.g., 2 open and 2 closed quadrants, and are allowed to explore for approximately 5 minutes. This paradigm exploits an approach-avoidance conflict between normal exploratory activity and an aversion to open spaces in rodents. This test measures anxiety levels and can be used to evaluate the effectiveness of anti- anxiolytic drugs. The time spent in open quadrants versus closed quadrants may be recorded automatically, with, for example, the placement of photobeams at each transition site.

The food avoidance test involves exposure to novel food and objectively measuring, for example, food intake and intake latency. The behaviors measured are statistically analyzed using standard statistical tests. (See, e.g., B. A. Campbell, et al., J. Comp. Physiol. Psychol. 67:15-22 (1969)).

The elevated plus-maze test comprises exposure to a maze, without sides, on a platform, the animal's behavior is objectively measured by counting the number of maze entries and maze learning. The behavior is statistically analyzed using standard statistical tests. (See, e.g., H. A. Baldwin, et al, Brain Res. Bull, 20:603-606 (1988)).

The stimulant-induced hyperactivity test involves injection of stimulant drugs (e.g., amphetamines, cocaine, PCP, and the like), and objectively measuring, for example, motor activity, social interactions, cognitive behavior. The animal's behaviors are statistically analyzed using standard statistical tests. (See, e.g., P. B. S. Clarke, et al, Psychopharmacology 96:511-520 (1988); P. Kuczenski, et al, J. Neuroscience 11:2703-2712 (1991)).

The self-stimulation test generally comprises providing the mouse with the opportunity to regulate electrical and/or chemical stimuli to its own brain. Behavior is measured by frequency and pattern of self-stimulation. Such behaviors are statistically analyzed using standard statistical tests.

(See, e.g., S. Nassif, et al, Brain Res., 332:247-257 (1985); W. L. Isaac, et al, Behav. Neurosci. 103:345-355 (1989)).

The reward test involves shaping a variety of behaviors, e.g., motor, cognitive, and social, measuring, for example, rapidity and reliability of behavioral change, and statistically analyzing the behaviors measured. (See, e.g., L. E. larrard, et al, Exp. Brain Res. 61:519-530 (1986)).

The DRL (differential reinforcement to low rates of responding) performance test involves exposure to intermittent reward paradigms and measuring the number of proper responses, e.g., lever pressing. Such behavior is statistically analyzed using standard statistical tests. (See, e.g.p. D. Sinden, et al, Behav. Neurosci. 100:320-329 (1986); V. Nalwa, et al, Behav Brain Res. 17:73-76 (1985); and

A. J. Nonneman, et al, J. Comp. Physiol. Psych. 95:588-602 (1981)).

The spatial learning test involves exposure to a complex novel environment, measuring the rapidity and extent of spatial learning, and statistically analyzing the behaviors measured. (See, e.g.,

N. Pitsikas, et al, Pharm. Bioch. Behav. 38:931-934 (1991); B. Poucet, et al, Brain Res. 37:269-280 (1990); D. Christie, et al, Brain Res. 37:263-268 (1990); and F. Van Haaren, et al, Behav. Neurosci. 102:481-488 (1988)). Alternatively, an open-field (of) test may be used, in which the greater distance traveled for a given amount of time is a measure of the activity level and anxiety of the animal. When the open field is a novel environment, it is believed that an approach-avoidance situation is created, in which the animal is "torn" between the drive to explore and the drive to protect itself. Because the chamber is lighted and has no places to hide other than the corners, it is expected that a "normal" mouse will spend more time in the corners and around the periphery than it will in the center where there is no place to hide. "Normal" mice will, however, venture into the central regions as they explore more and more of the chamber. It can then be extrapolated that especially anxious mice will spend most of their time in the corners, with relatively little or no exploration of the central region, whereas bold (i.e., less anxious) mice will travel a greater distance, showing little preference for the periphery versus the central region.

The visual, somatosensory and auditory neglect tests generally comprise exposure to a sensory stimulus, objectively measuring, for example, orientating responses, and statistically analyzing the behaviors measured. (See, e.g., I. M. Vargo, et al., Exp. Neurol. 102:199-209 (1988)).

The consummatory behavior test generally comprises feeding and drinking, and objectively measuring quantity of consumption. The behavior measured is statistically analyzed using standard statistical tests. (See, e.g., P. I. Fletcher, et al., Psychopharmacol. 102:301-308 (1990); M. G. Corda, et al.„ Proc. Nat'l Acad. Sci. USA 80:2072-2076 (1983)). A visual discrimination test can also be used to evaluate the visual processing of an animal.

One or two similar objects are placed in an open field and the animal is allowed to explore for about 5-10 minutes. The time spent exploring each object (proximity to, i.e., movement within, e.g., about 3-5 cm of the object is considered exploration of an object) is recorded. The animal is then removed from the open field, and the objects are replaced by a similar object and a novel object. The animal is returned to the open field and the percent time spent exploring the novel object over the old object is measured (again, over about a 5-10 minute span). "Normal" animals will typically spend a higher percentage of time exploring the novel object rather than the old object. If a delay is imposed between sampling and testing, the memory task becomes more hippocampal-dependent. If no delay is imposed, the task is more based on simple visual discrimination. This test can also be used for olfactory discrimination, in which the objects (preferably, simple blocks) can be sprayed or otherwise treated to hold an odor. This test can also be used to determine if the animal can make gustatory discriminations; animals that return to the previously eaten food instead of novel food exhibit gustatory neophobia.

A hot plate analgesia test can be used to evaluate an animal' s sensitivity to heat or painful stimuli. For example, a mouse can be placed on an approximately 55°C hot plate and the mouse's response latency (e.g., time to pick up and lick a hind paw) can be recorded. These responses are not reflexes, but rather "higher" responses requiring cortical involvement. This test may be used to evaluate a nociceptive disorder.

An accelerating rotarod test may be used to measure coordination and balance in mice. Animals can be, for example, placed on a rod that acts like a rotating treadmill (or rolling log). The rotarod can be made to rotate slowly at first and then progressively faster until it reaches a speed of, e.g., approximately 60 rpm. The mice must continually reposition themselves in order to avoid falling off. The animals are preferably tested in at least three trials, a minimum of 20 minutes apart. Those mice that are able to stay on the rod the longest are believed to have better coordination and balance. A metrazol administration test can be used to screen animals for varying susceptibilities to seizures or similar events. For example, a 5mg/ml solution of metrazol can be infused through the tail vein of a mouse at a rate of, e.g., approximately 0.375 ml/min. The infusion will cause all mice to experience seizures, followed by death. Those mice that enter the seizure stage the soonest are believed to be more prone to seizures. Four distinct physiological stages can be recorded: soon after the start of infusion, the mice will exhibit a noticeable "twitch", followed by a series of seizures, ending in a fmal tensing of the body known as "tonic extension", which is followed by death. Target Gene Products

The present invention further contemplates use of the target gene sequence to produce target gene products. Target gene products may include proteins that represent functionally equivalent gene products. Such an equivalent gene product may contain deletions, additions or substitutions of amino acid residues within the amino acid sequence encoded by the gene sequences described herein, but which result in a silent change, thus producing a functionally equivalent target gene product. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobi- city, hydrophilicity, and/or the amphipathic nature of the residues involved.

For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) • amino acids include aspartic acid and glutamic acid. "Functionally equivalent", as utilized herein, refers to a protein capable of exhibiting a substantially similar in vivo activity as the endogenous gene products encoded by the target gene sequences. Alternatively, when utilized as part of an assay, "functionally equivalent" may refer to peptides capable of interacting with other cellular or extracellular molecules in a manner substantially similar to the way in which the corresponding portion of the endogenous gene product would.

Other protein products useful according to the methods of the invention are peptides derived from or based on the target gene produced by recombinant or synthetic means (derived peptides). Target gene products may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing the gene polypeptides and peptides of the invention by expressing nucleic acid encoding gene sequences are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing gene protein coding sequences and appropriate transcriptional/translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination (see, e.g., Sambrook, et al., 1989, supra, and Ausubel, et al., 1989, supra). Alternatively, RNA capable of encoding gene protein sequences may be chemically synthesized using, for example, automated synthesizers (see, e.g. Oligonucleotide Synthesis: A Practical Approach, Gait, M. I. ed., IRL Press, Oxford (1984)).

A variety of host-expression vector systems may be utilized to express the gene coding sequences of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, exhibit the gene protein of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing gene protein coding sequences; yeast (e.g. Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the gene protein coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the gene protein coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing gene protein coding sequences; or mammalian cell systems (e.g. COS, CHO, BHK, 293, 3T3) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionine promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5 K promoter).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the gene protein being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of antibodies or to screen peptide libraries, for example, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al, EMBO J., 2:1791-94 (1983)), in which the gene protein coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res., 13:3101-09 (1985); Van Heeke et al, J. Biol. Chem., 264:5503-9 (1989)); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene protein can be released from the GST moiety. In a preferred embodiment, full length cDNA sequences are appended with in-frame Bam HI sites at the amino terminus and Eco RI sites at the carboxyl terminus using standard PCR methodologies (Innis, et al. (eds) PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego (1990)) and ligated into the pGEX-2TK vector (Pharmacia, Uppsala, Sweden). The resulting cDNA construct contains a kinase recognition site at the amino terminus for radioactive labeling and glutathione S-transferase sequences at the carboxyl terminus for affinity purification (Nilsson, et al, EMBO J., A: 1075-80 (1985); Zabeau et al, EMBO J., 1: 1217-24 (1982)).

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The gene coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of gene coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed (see, e.g., Smith, et al, J. Virol. 46: 584-93 (1983); U.S. Pat. No. 4,745,051).

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the gene coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing gene protein in infected hosts, (e.g., see Logan et al, Proc. Natl. Acad. Sci. USA, 81:3655-59 (1984)). Specific initiation signals may also be required for efficient translation of inserted gene coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of the gene coding sequence is inserted, exogenous translational control signals, including, perhaps, the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bitter, et al., Methods in Enzymol, 153:516-44 (1987)).

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the gene protein may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells which stably integrate the plasmid into their chromosomes and grow, to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the gene protein. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the gene protein. In a preferred embodiment, control of timing and/or quantity of expression of the recombinant protein can be controlled using an inducible expression construct. Inducible constructs and systems for inducible expression of recombinant proteins will be well known to those skilled in the art. Examples of such inducible promoters or other gene regulatory elements include, but are not limited to, tetracycline, metallothionine, ecdysone, and other steroid-responsive promoters, rapamycin responsive promoters, and the like (No, et al., Proc. Natl. Acad. Sci. USA, 93:3346-51 (1996); Furth, et al., Proc. Natl. Acad. Sci. USA, 91:9302-6 (1994)). Additional control elements that can be used include promoters requiring specific transcription factors such as viral, particularly HIV, promoters, hi one in embodiment, a Tet inducible gene expression system is utilized. (Gossen et al, Proc. Natl. Acad. Sci. USA, 89:5547-51 (1992); Gossen, et al, Science, 268: 1766-69 (1995)). Tet Expression Systems are based on two regulatory elements derived from the tetracycline-resistance operon of the E. coli TnlO transposon — the tetracycline repressor protein (TetR) and the tetracycline operator sequence (tetO) to which TetR binds. Using such a system, expression of the recombinant protein is placed under the control of the tetO operator sequence and transfected or transformed into a host cell. In the presence of TetR, which is co-transfected into the host cell, expression of the recombinant protein is repressed due to binding of the TetR protein to the tetO regulatory element. High-level, regulated gene expression can then be induced in response to varying concentrations of tetracycline (Tc) or Tc derivatives such as doxycycline (Dox), which compete with tetO elements for binding to TetR. Constructs and materials for tet inducible gene expression are available commercially from CLONTECH Laboratories, Inc., Palo Alto, CA.

When used as a component in an assay system, the gene protein may be labeled, either directly or indirectly, to facilitate detection of a complex formed between the gene protein and a test substance. Any of a variety of suitable labeling systems may be used including but not limited to radioisotopes such as 1251; enzyme labeling systems that generate a detectable calorimetric signal or light when exposed to substrate; and fluorescent labels. Where recombinant DNA technology is used to produce the gene protein for such assay systems, it may be advantageous to engineer fusion proteins that can facilitate labeling, immobilization and/or detection.

Indirect labeling involves the use of a protein, such as a labeled antibody, which specifically binds to the gene product. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library. Production of Antibodies Described herein are methods for the production of antibodies capable of specifically recognizing one or more epitopes. Such antibodies may include, but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. Such antibodies may be used, for example, in the detection of a target gene in a biological sample, or, alternatively, as a method for the inhibition of abnormal target gene activity. Thus, such antibodies may be utilized as part of disease treatment methods, and/or may be used as part of diagnostic techniques whereby patients may be tested for abnormal levels of target gene proteins, or for the presence of abnormal forms of such proteins. For the production of antibodies, various host animals may be immunized by injection with the target gene, its expression product or a portion thereof. Such host animals may include but are not limited to rabbits, mice, and rats, to name but a few. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Gueriή) and Corynebacterium parvum.

Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as target gene product, or an antigenic functional derivative thereof. For the production of polyclonal antibodies, host animals such as those described above, may be immunized by injection with gene product supplemented with adjuvants as also described above.

Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to the hybridoma technique of Kόhler and Milstein, Nature, 256:495-7 (1975); and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique (Kosbor, et al, Immunology Today, 4:72 (1983); Cote, et al, Proc. Natl. Acad. Sci. USA, 80:2026-30 (1983)), and the EBV-hybridoma technique (Cole, et al, in Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., New York, pp. 77-96 (1985)). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.

In addition, techniques developed for the production of "chimeric antibodies" (Morrison, et al, Proc. Natl. Acad. Sci., 81:6851-6855 (1984); Takeda, et al, Nature, 314:452-54 (1985)) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. Alternatively, techniques described for the production of single chain antibodies (U.S. Pat.

No. 4,946,778; Bird, Science 242:423-26 (1988); Huston, et al, Proc. Natl. Acad. Sci. USA, 85:5879- 83 (1988); and Ward, et al, Nature, 334:544-46 (1989)) can be adapted to produce gene-single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed (Huse, et al, Science, 246:1275-81 (1989)) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. Screening Methods

The present invention may be employed in a process for screening for agents such as agonists, i.e. agents that bind to and activate target gene polypeptides, or antagonists, i.e. inhibit the activity or interaction of target gene polypeptides with its ligand. Thus, polypeptides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures as known in the art. Any methods routinely used to identify and screen for agents that can modulate receptors may be used in accordance with the present invention.

The present invention provides methods for identifying and screening for agents that modulate target gene expression or function. More particularly, cells that contain and express target gene sequences may be used to screen for therapeutic agents. Such cells may include non- recombinant monocyte cell lines, such as U937 (ATCC# CRL-1593), THP-1 (ATCC# TIB-202), and P388D1 (ATCC# TIB-63); endothelial cells such as HUVEC's and bovine aortic endothelial cells (BAEC's); as well as generic mammalian cell lines such as HeLa cells and COS cells, e.g., COS-7 (ATCC# CRL-1651). Further, such cells may include recombinant, transgenic cell lines. For example, the transgenic mice of the invention may be used to generate cell lines, containing one or more cell types involved in a disease, that can be used as cell culture models for that disorder. While cells, tissues, and primary cultures derived from the disease transgenic animals of the invention may be utilized, the generation of continuous cell lines is preferred. For examples of techniques which may be used to derive a continuous cell line from the transgenic animals, see Small, et al., Mol. Cell Biol, 5:642-48 (1985).

Target gene sequences may be introduced into, and overexpressed in, the genome of the cell of interest. In order to overexpress a target gene sequence, the coding portion of the target gene sequence may be ligated to a regulatory sequence which is capable of driving gene expression in the cell type of interest. Such regulatory regions will be well known to those of skill in the art, and may be utilized in the absence of undue experimentation. Target gene sequences may also be disrupted or underexpressed. Cells having target gene disruptions or underexpressed target gene sequences may be used, for example, to screen for agents capable of affecting alternative pathways which compensate for any loss of function attributable to the disruption or underexpression. In vitro systems may be designed to identify compounds capable of binding the target gene products. Such compounds may include, but are not limited to, peptides made of D-and/or L-configu- ration amino acids (in, for example, the form of random peptide libraries; see e.g., Lam, et al, Nature, 354:82-4 (1991)), phosphopeptides (in, for example, the form of random or partially degenerate, directed phosphopeptide libraries; see, e.g., Songyang, et al, Cell, 12:161-18 (1993)), antibodies, and small organic or inorganic molecules. Compounds identified may be useful, for example, in modulating the activity of target gene proteins, preferably mutant target gene proteins; elaborating the biological function of the target gene protein; or screening for compounds that disrupt normal target gene interactions or themselves disrupt such interactions.

The principle of the assays used to identify compounds that bind to the target gene protein involves preparing a reaction mixture of the target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex which can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways. For example, one method to conduct such an assay would involve anchoring the target gene protein or the test substance onto a solid phase and detecting target protein/ test substance complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, the target gene protein may be anchored onto a solid surface, and the test compound, which is not anchored, may be labeled, either directly or indirectly.

In practice, microtitre plates are conveniently utilized. The anchored component may be immobilized by non-covalent or covalent attachments. Non-covalent attachment may be accomplished simply by coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody, specific for the protein may be used to anchor the protein to the solid surface. The surfaces may be prepared in advance and stored.

In order to conduct the assay, the nonimmobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously nonimmobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously nonimmobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the previously nonim- mobilized component (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for target gene product or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes.

Compounds that are shown to bind to a particular target gene product through one of the methods described above can be further tested for their ability to elicit a biochemical response from the target gene protein. Agonists, antagonists and/or inhibitors of the expression product can be identified utilizing assays well known in the art. Antisense, Ribozymes. and Antibodies

Other agents which may be used as therapeutics include the target gene, its expression product(s) and functional fragments thereof. Additionally, agents which reduce or inhibit mutant target gene activity may be used to ameliorate disease symptoms. Such agents include antisense, ribozyme, and triple helix molecules. Techniques for the production and use of such molecules are well known to those of skill in the art.

Anti-sense RNA and DNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation. With respect to antisense DNA, oligodeoxyribonucleotides derived from the translation initiation site, e.g., between the -10 and +10 regions of the target gene nucleotide sequence of interest, are preferred.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage. The composition of ribozyme molecules must include one or more sequences complementary to the target gene mRNA, and must include the well known catalytic sequence responsible for mRNA cleavage. For this sequence, see U.S. Pat. No. 5,093,246, which is incorporated by reference herein in its entirety. As such within the scope of the invention are engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of RNA sequences encoding target gene proteins. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the molecule of interest for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features, such as secondary structure, that may render the oligonucleotide sequence unsuitable. The suitability of candidate sequences may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.

Nucleic acid molecules to be used in triple helix formation for the inhibition of transcription should be single stranded and composed of deoxyribonucleotides. The base composition of these oligonucleotides must be designed to promote triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex. Nucleotide sequences may be pyrimidine-based, which will result in TAT and CGC triplets across the three associated strands of the resulting triple helix. The pyrimidine-rich molecules provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand. In addition, nucleic acid molecules may be chosen that are purine-rich, for example, containing a stretch of G residues. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in GGC triplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triple helix formation may be increased by creating a so called "switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5'-3', 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.

It is possible that the antisense, ribozyme, and/or triple helix molecules described herein may reduce or inhibit the transcription (triple helix) and or translation (antisense, ribozyme) of mRNA produced by both normal and mutant target gene alleles. In order to ensure that substantially normal levels of target gene activity are maintained, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal activity may be introduced into cells that do not contain sequences susceptible to whatever antisense, ribozyme, or triple helix treatments are being utilized. Alternatively, it may be preferable to coadminister normal target gene protein into the cell or tissue in order to maintain the requisite level of cellular or tissue target gene activity.

Anti-sense RNA and DNA, ribozyme, and triple helix molecules of the invention may be prepared by any method known in the art for the synthesis of DNA and RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines. Various well-known modifications to the DNA molecules may be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone. Antibodies that are both specific for target gene protein, and in particular, mutant gene protein, and interfere with its activity may be used to inhibit mutant target gene function. Such antibodies may be generated against the proteins themselves or against peptides corresponding to portions of the proteins using standard techniques known in the art and as also described herein. Such antibodies include but are not limited to polyclonal, monoclonal, Fab fragments, single chain antibodies, chimeric antibodies, etc. In instances where the target gene protein is intracellular and whole antibodies are used, internalizing antibodies may be preferred. However, lipofectin liposomes may be used to deliver the antibody or a fragment of the Fab region which binds to the target gene epitope into cells. Where fragments of the antibody are used, the smallest inhibitory fragment which binds to the target or expanded target protein's binding domain is preferred. For example, peptides having an amino acid sequence corresponding to the domain of the variable region of the antibody that binds to the target gene protein may be used. Such peptides may be synthesized chemically or produced via recombinant DNA technology using methods well known in the art (see, e.g., Creighton, Proteins: Structures and Molecular Principles (1984) W.H. Freeman, New York 1983, supra; and Sambrook, et al., 1989, supra). Alternatively, single chain neutralizing antibodies which bind to intracellular target gene epitopes may also be administered. Such single chain antibodies may be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population by utilizing, for example, techniques such as those described in Marasco, et al, Proc. Natl. Acad. Sci. USA, 90:7889-93 (1993). RNA sequences encoding target gene protein may be directly administered to a patient exhibiting disease symptoms, at a concentration sufficient to produce a level of target gene protein such that disease symptoms are ameliorated. Patients may be treated by gene replacement therapy. One or more copies of a normal target gene, or a portion of the gene that directs the production of a normal target gene protein with target gene function, may be inserted into cells using vectors which include, but are not limited to adenovirus, adeno-associated virus, and retrovirus vectors, in addition to other particles that introduce DNA into cells, such as liposomes. Additionally, techniques such as those described above may be utilized for the introduction of normal target gene sequences into human cells.

Cells, preferably, autologous cells, containing normal target gene expressing gene sequences may then be introduced or reintroduced into the patient at positions which allow for the amelioration of disease symptoms.

Pharmaceutical Compositions, Effective Dosages, and Routes of Administration The identified compounds that inhibit target mutant gene expression, synthesis and/or activity can be administered to a patient at therapeutically effective doses to treat or ameliorate the disease. A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of the disease.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅0 ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. Thus, the compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral, topical, subcutaneous, intraperitoneal, intravenous, intrapleural, intraoccular, intraarterial, or rectal administration. It is also contemplated that pharmaceutical compositions may be administered with other products that potentiate the activity of the compound and optionally, may include other therapeutic ingredients.

For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated to give controlled release of the active compound.

For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. Oral ingestion is possibly the easiest method of taking any medication. Such a route of administration, is generally simple and straightforward and is frequently the least inconvenient or unpleasant route of administration from the patient's point of view. However, this involves passing the material through the stomach, which is a hostile environment for many materials, including proteins and other biologically active compositions. As the acidic, hydrolytic and proteolytic environment of the stomach has evolved efficiently to digest proteinaceous materials into amino acids and oligopeptides for subsequent anabolism, it is hardly surprising that very little or any of a wide variety of biologically active proteinaceous material, if simply taken orally, would survive its passage through the stomach to be taken up by the body in the small intestine. The result, is that many proteinaceous medicaments must be taken in through another method, such as parenterally, often by subcutaneous, intramuscular or intravenous injection.

Pharmaceutical compositions may also include various buffers (e.g., Tris, acetate, phosphate), solubilizers (e.g., Tween, Polysorbate), carriers such as human serum albumin, preservatives (thimerosol, benzyl alcohol) and anti-oxidants such as ascorbic acid in order to stabilize pharmaceu- tical activity. The stabilizing agent may be a detergent, such as tween-20, tween-80, NP-40 or Triton X-100. EBP may also be incorporated into particulate preparations of polymeric compounds for controlled delivery to a patient over an extended period of time. A more extensive survey of components in pharmaceutical compositions is found in Remington's Pharmaceutical Sciences, 18th ed., A. R. Gennaro, ed., Mack Publishing, Easton, Pa. (1990).

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. Diagnostics A variety of methods may be employed to diagnose disease conditions associated with the target gene. Specifically, reagents may be used, for example, for the detection of the presence of target gene mutations, or the detection of either over or under expression of target gene mRNA.

According to the diagnostic and prognostic method of the present invention, alteration of the wild-type target gene locus is detected. In addition, the method can be performed by detecting the wild-type target gene locus and confirming the lack of a predisposition or neoplasia. "Alteration of a wild-type gene" encompasses all forms of mutations including deletions, insertions and point mutations in the coding and noncoding regions. Deletions may be of the entire gene or only a portion of the gene. Point mutations may result in stop codons, frameshift mutations or amino acid substitutions. Somatic mutations are those which occur only in certain tissues, e.g., in the tumor tissue, and are not inherited in the germline. Germline mutations can be found in any of a body's tissues and are inherited. If only a single allele is somatically mutated, an early neoplastic state is indicated. However, if both alleles are mutated, then a late neoplastic state may be indicated. The finding of gene mutations thus provides both diagnostic and prognostic information. A target gene allele which is not deleted (e.g., that found on the sister chromosome to a chromosome carrying a target gene deletion) can be screened for other mutations, such as insertions, small deletions, and point mutations. Mutations found in tumor tissues may be linked to decreased expression of the target gene product. However, mutations leading to non-functional gene products may also be linked to a cancerous state. Point mutational events may occur in regulatory regions, such as in the promoter of the gene, leading to loss or diminution of expression of the mRNA. Point mutations may also abolish proper RNA processing, leading to loss of expression of the target gene product, or a decrease in mRNA stability or translation efficiency. One test available for detecting mutations in a candidate locus is to directly compare genomic target sequences from cancer patients with those from a control population. Alternatively, one could sequence messenger RNA after amplification, e.g., by PCR, thereby eliminating the necessity of determining the exon structure of the candidate gene. Mutations from cancer patients falling outside the coding region of the target gene can be detected by examining the non-coding regions, such as introns and regulatory sequences near or within the target gene. An early indication that mutations in noncoding regions are important may come from Northern blot experiments that reveal messenger

RNA molecules of abnormal size or abundance in cancer patients as compared to control individuals.

The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one specific gene nucleic acid or anti-gene antibody reagent described herein, which may be conveniently used, e.g., in clinical settings, to diagnose patients exhibiting disease symptoms or at risk for developing disease.

Any cell type or tissue, preferably monocytes, endothelial cells, or smooth muscle cells, in which the gene is expressed may be utilized in the diagnostics described below. DNA or RNA from the cell type or tissue to be analyzed may easily be isolated using procedures which are well known to those in the art. Diagnostic procedures may also be performed in situ directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. Nucleic acid reagents may be used as probes and or primers for such in situ procedures (see, for example, Nuovo, PCR In Situ Hybridization: Protocols and Applications, Raven Press, N.Y. (1992)).

Gene nucleotide sequences, either RNA or DNA, may, for example, be used in hybridization or amplification assays of biological samples to detect disease-related gene structures and expression. Such assays may include, but are not limited to, Southern or Northern analyses, restriction fragment length polymorphism assays, single stranded conformational polymorphism analyses, in situ hybridization assays, and polymerase chain reaction analyses. Such analyses may reveal both quantitative aspects of the expression pattern of the gene, and qualitative aspects of the gene expression and or gene composition. That is, such aspects may include, for example, point mutations, insertions, deletions, chromosomal rearrangements, and or activation or inactivation of gene expression. Preferred diagnostic methods for the detection of gene-specific nucleic acid molecules may involve for example, contacting and incubating nucleic acids, derived from the cell type or tissue being analyzed, with one or more labeled nucleic acid reagents under conditions favorable for the specific annealing of these reagents to their complementary sequences within the nucleic acid molecule of interest. Preferably, the lengths of these nucleic acid reagents are at least 9 to 30 nucleotides. After incubation, all non-annealed nucleic acids are removed from the nucleic acid:fingerprint molecule hybrid. The presence of nucleic acids from the fingerprint tissue which have hybridized, if any such molecules exist, is then detected. Using such a detection scheme, the nucleic acid from the tissue or cell type of interest may be immobilized, for example, to a solid support such as a membrane, or a plastic surface such as that on a microtitre plate or polystyrene beads. In this case, after incubation, non-annealed, labeled nucleic acid reagents are easily removed. Detection of the remaining, annealed, labeled nucleic acid reagents is accomplished using standard techniques well-known to those in the art.

Alternative diagnostic methods for the detection of gene-specific nucleic acid molecules may involve their amplification, e.g., by PCR (the experimental embodiment set forth in Mullis U.S. Pat. No. 4,683,202 (1987)), ligase chain reaction (Barany, Proc. Natl. Acad. Sci. USA, 88:189-93 (1991)), self sustained sequence replication (Guatelli, et al, Proc. Natl. Acad. Sci. USA, 87:1874-78 (1990)), transcriptional amplification system (Kwoh, et al, Proc. Natl. Acad. Sci. USA, 86: 1173-77 (1989)), Q-Beta Replicase (Lizardi et al, Bio/Technology, 6:1197 (1988)), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

In one embodiment of such a detection scheme, a cDNA molecule is obtained from an RNA molecule of interest (e.g., by reverse transcription of the RNA molecule into cDNA). Cell types or tissues from which such RNA may be isolated include any tissue in which wild type fingerprint gene is known to be expressed, including, but not limited, to monocytes, endothelium, and/or smooth muscle. A sequence within the cDNA is then used as the template for a nucleic acid amplification reaction, such as a PCR amplification reaction, or the like. The nucleic acid reagents used as synthesis initiation reagents (e.g., primers) in the reverse transcription and nucleic acid amplification steps of this method may be chosen from among the gene nucleic acid reagents described herein. The preferred lengths of such nucleic acid reagents are at least 15-30 nucleotides. For detection of the amplified product, the nucleic acid amplification may be performed using radioactively or non- radioactively labeled nucleotides. Alternatively, enough amplified product may be made such that the product may be visualized by standard ethidium bromide staining or by utilizing any other suitable nucleic acid staining method.

Antibodies directed against wild type or mutant gene peptides may also be used as disease diagnostics and prognostics. Such diagnostic methods, may be used to detect abnormalities in the level of gene protein expression, or abnormalities in the structure and/or tissue, cellular, or subcellular location of fingerprint gene protein. Structural differences may include, for example, differences in the size, electronegativity, or antigenicity of the mutant fingerprint gene protein relative to the normal fingerprint gene protein. Protein from the tissue or cell type to be analyzed may easily be detected or isolated using techniques which are well known to those of skill in the art, including but not limited to western blot analysis. For a detailed explanation of methods for carrying out western blot analysis. (See, e.g. Sambrook, et al. (1989) supra, at Chapter 18). The protein detection and isolation methods employed herein may also be such as those described in Harlow and Lane, for example, (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988)). Preferred diagnostic methods for the detection of wild type or mutant gene peptide molecules may involve, for example, immunoassays wherein fingerprint gene peptides are detected by their interaction with an anti-fingerprint gene-specific peptide antibody.

For example, antibodies, or fragments of antibodies useful in the present invention may be used to quantitatively or qualitatively detect the presence of wild type or mutant gene peptides. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently

^■ labeled antibody (see below) coupled with light microscopic, flow cytometric, or fluorimetric detection. Such techniques are especially preferred if the fingerprint gene peptides are expressed on the cell surface.

The antibodies (or fragments thereof) useful in the present invention may, additionally, be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of fingerprint gene peptides. In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody of the present invention. The antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the fingerprint gene peptides, but also their distribution in the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.

Immunoassays for wild type, mutant, or expanded fingerprint gene peptides typically comprise incubating a biological sample, such as a biological fluid, a tissue extract, freshly harvested cells, or cells which have been incubated in tissue culture, in the presence of a detectably labeled antibody capable of identifying fingerprint gene peptides, and detecting the bound antibody by any of a number of techniques well known in the art.

The biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled gene-specific antibody. The solid phase support may then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on solid support may then be detected by conventional means.

The terms "solid phase support or carrier" are intended to encompass any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacryla- mides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.

The binding activity of a given lot of anti-wild type or -mutant fingerprint gene peptide antibody may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.

One of the ways in which the gene peptide-specific antibody can be detectably labeled is by linking the same to an enzyme and using it in an enzyme immunoassay (EIA) (Voller, Rie Clin Lab, 8:289-98 (1978) ["The Enzyme Linked Immunosorbent Assay (ELISA)", Diagnostic Horizons 2:1-7, 1978, Microbiological Associates Quarterly Publication, Walkersville, Md.]; Voller, et al, J. Clin. PathoL, 31:507-20 (1978); Butler, Meth. Enzymol, 73:482-523 (1981); Maggio (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla. (1980); Ishikawa, et al, (eds.) Enzyme Immunoassay, Igaku-Shoin, Tokyo (1981)). The enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means. Enzymes which can be used to detectably label the antibody include,, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards. Detection may also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect fingerprint gene wild type, mutant, or expanded peptides through the use of a radioimmunoassay (RIA) (see, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986). The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography. It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTP A) or ethylenediamine-tetraacetic acid (EDTA). The antibody also can be detectably labeled by coupling it to a chemiluminescent compound.

The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin. Throughout this application, various publications, patents and published patent applications are referred to by an identifying citation. The disclosures of these publications, patents and published patent specifications referenced in this application are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.

The following examples are intended only to illustrate certain aspects and embodiments of the present invention and should in no way be construed as limiting the subject invention.

Examples

Example 1: Generation and Analysis of Mice Comprising DEZ Orphan GPCR Gene Disruptions

Targeting Construct for DEZ Orphan GPCR Gene. To investigate the role of the DEZ recep- tor gene, disruptions in genes comprising the sequence set forth in SEQ ID NO:l (see Figure 1) were produced by homologous recombination. More particularly, as shown in Figures 2 and 3, a specific targeting construct having the ability to disrupt or modify genes comprising SEQ ID NO: l was created, using as the targeting arms (homologous sequences) in the construct the sequences identified herein as SEQ ID NO:2 and SEQ ID NO:3 (see Figure 3). Transgenic Mice. The targeting construct was introduced into ES cells by electroporation and chimeric mice were generated. The ES cells were derived from 129/Sv-+P+Mgf-SLJ/J mouse substrain. FI mice were generated by breeding with C57BL/6 females. The resultant FINO heterozygotes were intercrossed to produce F2N0 homozygotes, or were backcrossed to C57BL/6 mice to generate F1N1 heterozygotes. F2N1 homozygous mutant mice were produced by intercrossing F1N1 heterozygous males and females.

Phenotypic Analysis. The transgenic mice were analyzed for phenotypic changes. When compared to age- and gender-matched wild-type control mice, homozygous mutant mice exhibited decreased agility or coordination, as characterized by decreased performance (i.e., decreased latency) on an accelerating rotarod, i.e., the homozygous mutants fell from the rod at a lower speed than their wild-type counterparts, which increased their latency to fall across three consecutive trials (see Table

1).

TABLE 1 - ROTAROD FALL SPEED

Example 2: Generation and Analysis of Mice Comprising CB2R Gene Disruptions

Targeting Construct for CB2R Gene. To investigate the role of genes encoding cannabinoid receptors, particularly CB2R receptor genes, disruptions in genes comprising the sequence set forth in SEQ ID NO: 4 (see Figure 4) were produced by homologous recombination. More particularly, as shown in Figures 5 and 6, a specific targeting construct having the ability to disrupt or modify genes comprising SEQ ID NO:4 was created, using as the targeting arms (homologous sequences) in the construct the sequences identified herein as SEQ ID NO:5 and SEQ ID NO:6 (see Figure 6). Transgenic Mice. The targeting construct was introduced into ES cells derived from the

129/OlaHsd mouse substrain by electroporation to generate chimeric mice. FI mice were generated by breeding with C57BL/6 females. The resultant F1N0 heterozygotes were intercrossed to produce F2N0 homozygotes, or were backcrossed to C57BL/6 mice to generate F1N1 heterozygotes. F2N1 homozygous mutant mice were produced by intercrossing F1N1 heterozygous males and females. Example 3: Generation and Analysis of Mice Comprising Adrenomedullin Receptor Gene Disruptions

Targeting Construct. To investigate the role of genes encoding adrenomedullin receptors, disruptions in genes comprising the sequence set forth in SEQ ID NO:7 (see Figure 7) were produced by homologous recombination. More particularly, as shown in Figures 8 and 9, a specific targeting construct having the ability to disrupt or modify genes comprising SEQ ID NO:7 was created, using as the targeting arms (homologous sequences) in the construct the sequences identified herein as SEQ ID NO:8 and SEQ ID NO:9 (see Figure 9).

Transgenic Mice. The targeting construct was introduced into ES cells derived from the 129/OlaHsd mouse substrain by electroporation to generate chimeric mice. FI mice were generated by breeding with C57BL/6 females. The resultant F1N0 heterozygotes were intercrossed to produce F2N0 homozygotes, or were backcrossed to C57BL/6 mice to generate F1N1 heterozygotes. F2N1 homozygous mutant mice were produced by intercrossing F1N1 heterozygous males and females.

Phenotypic Analysis. The transgenic mice were analyzed for phenotypic changes. When compared to age- and gender-matched wild-type control mice, homozygous mutant mice displayed decreases in average velocity, total distance traveled (see Table 2) and percentage of time spent in the central region (see Table 3) during Open Field testing, indicating that the homozygotes exhibit decreased activity (e.g., hypoactivity) and increased anxiety. TABLE 2 - TOTAL DISTANCE TRAVELED IN OPEN FIELD TEST

TABLE 3 - PERCENTAGE OF TIME IN CENTRAL REGION OF OPEN FIELD

Example 4: Generation and Analysis of Mice Comprising CRFR2 Gene Disruptions

Targeting Construct. To investigate the role of CRFR2 genes, disruptions in genes comprising the sequence set forth in SEQ ID NO: 10 (Figure 10) were produced by homologous recombination. More particularly, as shown in Figure 11 and 12, a specific targeting construct having the ability to disrupt or modify genes comprising SEQ ID NO: 10 was created, using as the targeting arms (homologous sequences) in the construct the sequences identified herein as SEQ ID NO: 11 and SEQ ID NO: 12 (see Figure 12). Transgenic Mice. The targeting construct was introduced into ES cells derived from the 129/SvJ x 129/Sv-CP mouse by electroporation to generate chimeric mice. FI mice were generated by breeding with C57BL/6 females. The resultant FINO heterozygotes were intercrossed to produce F2N0 homozygotes, or were backcrossed to C57BL/6 mice to generate FlNl heterozygotes. F2N1 homozygous mutant mice were produced by intercrossing FlNl heterozygous males and females.

Phenotypic Analysis. The transgenic mice were analyzed for phenotypic changes. When compared to age- and gender-matched wild-type mice, homozygous mutant mice displayed decreased activity, as evidenced by total distance traveled in an open field test (see Table 4) and appeared hypoactive. The homozygotes also exhibited a decreased susceptibility to seizure, as evidenced by an increased metrazol response threshold, i.e., homozygotes required a higher dose of metrazol to show the stereotypical seizure responses (first twitch; tonic/clonic (T/C) seizure; tonic extension; and death), particularly dose to tonic extension and dose to respiratory arrest or death (see Table 5). TABLE 4 - TOTAL DISTANCE TRAVELED IN OPEN FIELD TEST

TABLE 5 - METRAZOL DOSE TEST

Example 5: Generation and Analysis of Mice Comprising FPRL3 N-Formylpeptide Receptor Gene Disruptions

Targeting Construct for FPRL3 Gene. To investigate the role of genes encoding N-formylpeptide receptors, particularly FPRL3 genes, disruptions in genes comprising the sequence set forth in SEQ ID NO: 13 (see Figure 13) were produced by homologous recombination. More particularly, as shown in Figures 14 and 15, a specific targeting construct having the ability to disrupt or modify genes comprising SEQ ID NO: 13 was created, using as the targeting arms (homologous sequences) in the construct the sequences identified herein as SEQ ID NO: 14 and SEQ ID NO: 15 (see Figure 15).

Transgenic Mice. The targeting construct was introduced into ES cells derived from the 129/OlaHsd mouse substrain by electroporation to generate chimeric mice. FI mice were generated by breeding with C57BL/6 females. The resultant FINO heterozygotes were intercrossed to produce F2N0 homozygotes, or were backcrossed to C57BL/6 mice to generate FlNl heterozygotes. F2N1 homozygous mutant mice were produced by intercrossing FlNl heterozygous males and females. Example 6: Generation and Analysis of Mice Comprising NPY4-R Gene Disruptions Targeting Construct. To investigate the role of NPY4-R genes, disruptions in genes comprising the sequence set forth in SEQ ID NO: 16 (see Figure 16) were produced by homologous recombination. More particularly, as shown in Figures 17 and 18, a specific targeting construct having the ability to disrupt or modify genes comprising SEQ ID NO: 16 was created, using as the targeting arms (homologous sequences) in the construct the sequences identified herein as SEQ ID NO: 17 and SEQ ID NO: 18 (see Figure 18).

Transgenic Mice. The targeting construct was introduced into ES cells derived from 129/OlaHsd mouse substrain by electroporation to generate chimeric mice. FI mice were generated by breeding with C57BL/6 females. The resultant FINO heterozygotes were intercrossed to produce F2N0 homozygotes, or were backcrossed to C57BL/6 mice to generate FlNl heterozygotes. F2N1 homozygous mutant mice were produced by intercrossing FlNl heterozygous males and females. Example 7: Generation and Analysis of Mice Comprising NPY6-R Gene Disruptions

Targeting Construct. To investigate the role of NPY6-R genes, disruptions in genes comprising the sequence set forth in SEQ ID NO: 19 (see Figure 19) were produced by homologous recombination. More particularly, as shown in Figures 20 and 21, a specific targeting construct having the ability to disrupt or modify genes comprising SEQ ID NO: 19 was created, using as the targeting arms (homologous sequences) in the construct the sequences identified herein as SEQ ID NO:20 and SEQ ID NO:21 (see Figure 21).

Transgenic Mice. The targeting construct was introduced into ES cells derived from 129/OlaHsd mouse substrain by electroporation to generate chimeric mice. FI mice were generated by breeding with C57BL/6 females. The resultant F1N0 heterozygotes were intercrossed to produce F2N0 homozygotes, or were backcrossed to C57BL/6 mice to generate FlNl heterozygotes. F2N1 homozygous mutant mice were produced by intercrossing FlNl heterozygous males and females.

Phenotypic Analysis. The transgenic mice were analyzed for phenotypic changes. When compared to age- and gender-matched wild-type control mice, homozygous mutant mice displayed enhanced performance during rotarod testing, i.e., the mean fall speed for mutant mice was increased for each of the trials tested, compared to wild-type littermates, suggesting that the homozygous mice exhibited increased agility or coordination, as characterized by increased performance (i.e., increased latency) on the accelerating rotarod (see Table 6). TABLE 6 - ROTAROD FALL SPEED

Example 8: Generation and Analysis of Mice Comprising KOR3 Gene Disruptions

Targeting Construct. To investigate the role of KOR3 genes, disruptions in genes comprising the sequence set forth in SEQ ID NO: 22 (see Figure 22) were produced by homologous recombination. More particularly, as shown in Figures 23 and 24, a specific targeting construct having the ability to disrupt or modify genes comprising SEQ ID NO: 22 was created, using as the targeting arms (homologous sequences) in the construct the sequences identified herein as SEQ ID NO:23 and SEQ ID NO:24 (see Figure 24).

Transgenic Mice. The targeting construct was introduced into ES cells derived from 129/OlaHsd mouse substrain by electroporation to generate chimeric mice. FI mice were generated by breeding with C57BL/6 females. The resultant F1N0 heterozygotes were intercrossed to produce F2N0 homozygotes, or were backcrossed to C57BL/6 mice to generate FlNl heterozygotes. F2N1 homozygous mutant mice were produced by intercrossing FlNl heterozygous males and females. Example 9: Generation and Analysis of Mice Comprising mGluR8 Gene Disruptions

Targeting Construct. To investigate the role of mGluR8 genes, disruptions in genes comprising the sequence set forth in SEQ ID NO:25 (see Figure 25) were produced by homologous recombination. More particularly, as shown in Figures 26 and 27, a specific targeting construct having the ability to disrupt or modify genes comprising SEQ ID NO: 25 was created, using as the targeting arms (homologous sequences) in the construct the sequences identified herein as SEQ ID NO:26 and SEQ ID NO:27 (see Figure 27). Trans genie Mice. The targeting construct was introduced into ES cells derived from 129/OlaHsd mouse substrain by electroporation to generate chimeric mice. FI mice were generated by breeding with C57BL/6 females. The resultant F1N0 heterozygotes were intercrossed to produce F2N0 homozygotes, or were backcrossed to C57BL/6 mice to generate FlNl heterozygotes. F2N1 homozygous mutant mice were produced by intercrossing FlNl heterozygous males and females.

Phenotypic Analysis. The transgenic mice were analyzed for phenotypic changes. Homozygous mutant mice exhibited an increase in total distance traveled in an open-field test relative to wild- type control mice (see Table 7), suggesting that the mutant mice are more active (e.g., hyperactive). TABLE 7 - TOTAL DISTANCE TRAVELED IN OPEN FIELD TEST

Homozygous mutant animals also displayed a statistically significant decrease in Pre-Pulse Inhibition (PPI) with a lOOdB pre-pulse. Mutants displayed significantly decreased PPI, indicating a neuropsychological disorder, particularly a stimulus-processing deficit similar to that observed in schizophrenic patients. These tests are described in more detail as follows:

Startle Test:

The startle test screens for changes in the basic fundamental nervous system or muscle-related functions. The startle reflex is a short-latency response of the skeletal musculature elicited by a sudden auditory stimulus. This includes changes in 1) hearing - auditory processing; 2) sensory and motor processing - related to the auditory circuit and culminating in a motor related output; 3) global sensory changes; and motor abnormalities, including skeletal muscle or motor neuron related changes.

The startle test also screens for higher-level cognitive functions. One component of the startle reflex test is prepulse inhibition (PPI). PPI is the attenuation of the startle reflex response produced by a "prepulse" stimulus. Deficits in PPI are observed in human schizophrenic patients. However, changes in the basic startle reflex in the absence of changes in PPI could also reflect higher-level cognitive changes. The startle reflex and PPI can be modulated by negative affective states like fear or stress. The cognitive changes include: 1) sensorimotor processing such as sensorimotor gating changes related to schizophrenia; 2) attention disorders; 3) anxiety disorders; and 4) thought disturbance disorders.

The homozygous mice were tested as follows:

Sound Response Profile: The mice were tested in a San Diego Instruments SR-LAB sound response chamber. Each mouse was exposed to 9 stimulus types that were repeated in pseudo-random order ten times during the course of the entire 25-minute test. The stimulus types in decibels were: p80, p90, plOO, pi 10, pl20, ρp80, pl20, pp90, pl20, pplOO, and pl20; where p=40 msec pulse, pp=20 msec prepulse The length of time between a prepulse and a pulse was 100 msec (onset to onset). The mean Vmax of the ten repetitions for each trial type was computed for each mouse. Pre-Pulse Inhibition: The % prepulse inhibition (PPI) compared to pl20 alone was computed for each mouse at three prepulse levels from the mean Vmax values. This was computed by determining the mean "pl20", "pp80pl20", "pp90pl20", and "ppl00pl20" value for each mouse and then producing the ratios of % inhibition. Example 10: Generation and Analysis of Mice Comprising MAS Gene Disruptions Targeting Construct. To investigate the role of MAS genes, disruptions in genes comprising the sequence set forth in SEQ ID NO:28 (see Figure 28) were produced by homologous recombination. More particularly, as shown in Figures 29 and 30, a specific targeting construct having the ability to disrupt or modify genes comprising SEQ ID NO: 28 was created, using as the targeting arms (homologous sequences) in the construct the sequences identified herein as SEQ ID NO:29 and SEQ ID NO:30 (see Figure 30).

Phenotypic Analysis. The transgenic mice were analyzed for phenotypic changes. When compared to age- and gender-matched wild-type control mice, homozygous mutants spent a decreased percentage of time in the central region of the open field (see Table 8), suggesting increased anxiety as compared to wild-types. Homozygous mutants also expelled significantly more fecal boli during the 10-minute open field test than their wild-type littermates, also suggesting an increased anxiety phenotype. Homozygous mutants also exhibited decreased activity (e.g., hypoactivity), traveling less total distance during the open field test than the wild-type mice (see Table 9). Homozygous mutants also exhibited a decreased susceptibility to seizure as evidenced by an increased response threshold to metrazol, i.e., they required a larger dose of metrazol to display several characteristic seizure response parameters, as compared to wild-type animals (see Table 10). These characteristics response parameters are: (1) first twitch; (2) tonic/clonic seizure; (3) tonic extension, or T/C; and (4) death. TABLE 8 - PERCENTAGE OF TIME IN CENTRAL REGION OF OPEN FIELD

TABLE 9 - TOTAL DISTANCE TRAVELED IN OPEN FIELD TEST

TABLE 10 - METRAZOL DOSE TESTING

As is apparent to one of skill in the art, various modifications of the above embodiments can be made without departing from the spirit and scope of this invention. These modifications and variations are within the scope of this invention.

Claims

We claim:

1. A targeting construct comprising:

(a) a first polynucleotide sequence homologous to a target gene selected from the group consisting of a DEZ receptor gene, a CB2R gene, an adrenomedullin receptor gene, a CRFR2 gene, an FPRL3 gene, an NPY4-R gene, an NPY6-R gene, a KOR3 gene, an mGluR8 gene, and a MAS receptor gene;

(b) a second polynucleotide sequence homologous to the target gene; and

(c) a selectable marker.

2. The targeting construct of claim 1, wherein the targeting construct further comprises a screening marker.

3. A method of producing a targeting construct for a target gene selected from the group consisting of a DEZ receptor gene, a CB2R gene, an adrenomedullin receptor gene, a CRFR2 gene, an FPRL3 gene, an NPY4-R gene, an NPY6-R gene, a KOR3 gene, an nιGluR8 gene, and a MAS receptor gene, the method comprising:

(a) obtaining a first polynucleotide sequence homologous to the target gene;

(b) obtaining a second polynucleotide sequence homologous to the target gene;

(c) providing a vector comprising a selectable marker; and

(d) inserting the first and second sequences into the vector, to produce the targeting construct.

4. A method of producing a targeting construct for a target gene selected from the group consisting of a DEZ receptor gene, a CB2R gene, an adrenomedullin receptor gene, a CRFR2 gene, an FPRL3 gene, an NPY4-R gene, an NPY6-R gene, a KOR3 gene, an mGluR8 gene, and a MAS receptor gene, the method comprising:

(a) providing a polynucleotide comprising a first sequence homologous to a first region of the target gene and a second sequence homologous to a second region of the target gene; and

(b) inserting a positive selection marker between the first and second sequences to form the targeting construct.

5. A cell comprising a disruption in a target gene selected from the group consisting of a DEZ receptor gene, a CB2R gene, an adrenomedullin receptor gene, a CRFR2 gene, an FPRL3 gene, an NPY4-R gene, an NPY6-R gene, a KOR3 gene, an mGluR8 gene, and a MAS receptor gene.

6. The cell of claim 5, wherein the cell is a murine cell.

7. The cell of claim 6, wherein the murine cell is an embryonic stem cell.

8. A non-human transgenic animal comprising a disruption in a target gene selected from the group consisting of a DEZ receptor gene, a CB2R gene, an adrenomedullin receptor gene, a CRFR2 gene, an FPRL3 gene, an NPY4-R gene, an NPY6-R gene, a KOR3 gene, an mGluR8 gene, and a MAS receptor gene.

9. A cell derived from the non-human transgenic animal of claim 8.

10. A method of producing a transgenic mouse, the method comprising:

(a) introducing the targeting construct of claim 1 into a cell;

(b) introducing the cell into a blastocyst;

(c) implanting the resulting blastocyst into a pseudopregnant mouse, wherein said pseudopregnant mouse gives birth to a chimeric mouse; and

(d) breeding the chimeric mouse to produce the transgenic mouse.

11. A method of identifying an agent that modulates the expression of a target gene selected from the group consisting of a DEZ receptor gene, a CB2R gene, an adrenomedullin receptor gene, a CRFR2 gene, an FPRL3 gene, an NPY4-R gene, an NPY6-R gene, a KOR3 gene, an mGluR8 gene, and a MAS receptor gene, the method comprising:

(a) providing a non-human transgenic animal comprising a disruption in the target gene;

(b) administering an agent to the non-human transgenic animal; and

(c) determining whether the expression of the disrupted target gene in the non-human transgenic animal is modulated.

12. A method of identifying an agent that modulates the function of a target gene selected from the group consisting of a DEZ receptor gene, a CB2R gene, an adrenomedullin receptor gene, a CRFR2 gene, an FPRL3 gene, an NPY4-R gene, an NPY6-R gene, a KOR3 gene, an mGluR8 gene, and a MAS receptor gene, the method comprising:

(b) administering an agent to the non-human transgenic animal; and

(c) determining whether the function of the disrupted target gene in the non-human transgenic animal is modulated.

13. A method of identifying an agent that modulates the expression of a target gene selected from the group consisting of a DEZ receptor gene, a CB2R gene, an adrenomedullin receptor gene, a CRFR2 gene, an FPRL3 gene, an NPY4-R gene, an NPY6-R gene, a KOR3 gene, an mGluR8 gene, and a MAS receptor gene, the method comprising:

(a) providing a cell comprising a disruption in the target gene;

Ob) contacting the cell with an agent; and

(c) determining whether expression of the target gene is modulated.

14. A method of identifying an agent that modulates the function of a target gene selected from the group consisting of a DEZ receptor gene, a CB2R gene, an adrenomedullin receptor gene, a CRFR2 gene, an FPRL3 gene, an NPY4-R gene, an NPY6-R gene, a KOR3 gene, an mGluR8 gene, and a MAS receptor gene, the method comprising:

(a) providing a cell comprising a disruption in the target gene;

(b) contacting the cell with an agent; and

(c) determining whether the function of the target gene is modulated.

15. The method of claim 13 or claim 14, wherein the cell is derived from the non-human transgenic animal of claim 8.

16. An agent identified by the method of claim 11, claim 12, claim 13, or claim 14.

17. A transgenic mouse comprising a homozygous disruption in a DEZ receptor gene.

18. The transgenic mouse of claim 17, wherein the transgenic mouse exhibits decreased agility or coordination relative to a wild-type mouse.

19. The transgenic mouse of claim 18, wherein the decreased agility or coordination is characterized by decreased latency in an accelerating rotarod test.

20. A method of identifying an agent that modulates a phenotype associated with the transgenic mouse of claim 17, the method comprising:

(a) administering an agent to the transgenic mouse; and

(b) determining whether the agent modulates the phenotype.

21. An agent identified by the method of claim 20.

22. An agonist or antagonist of a DEZ receptor.

23. A cell derived from the transgenic mouse of claim 17.

24. Phenotypic data associated with the transgenic mouse of claim 17, wherein the data is in a database.

25. A transgenic mouse comprising a homozygous disruption in an adrenomedullin receptor gene.

26. The transgenic mouse of claim 25, wherein the transgenic mouse exhibits decreased activity relative to a wild-type mouse.

27. The transgenic mouse of claim 26, wherein the transgenic mouse is hypoactive.

28. The transgenic mouse of claim 26, wherein the decreased activity is characterized by reduced distance traveled in an open field test.

29. The transgenic mouse of claim 25, wherein the transgenic mouse exhibits increased anxiety relative to a wild-type mouse.

30. The transgenic mouse of claim 27, wherein the increased anxiety is characterized by reduced percentage of time spent in the central region of an open field test.

31. A method of identifying an agent that modulates a phenotype associated with the transgenic mouse of claim 25, the method comprising:

(a) administering an agent to the transgenic mouse; and

(b) determining whether the agent modulates the phenotype.

32. An agent identified by the method of claim 31.

33. An agonist or antagonist of an adrenomedullin receptor.

34. A cell derived from the transgenic mouse of claim 25.

35. Phenotypic data associated with the transgenic mouse of claim 25, wherein the data is in a database.

36. A transgenic mouse comprising a homozygous disruption in a CRFR2 gene.

37. The transgenic mouse of claim 36, wherein the transgenic mouse exhibits decreased activity relative to a wild-type mouse.

38. The transgenic mouse of claim 37, wherein the transgenic mouse is hypoactive.

39. The transgenic mouse of claim 37, wherein the decreased activity is characterized by reduced distance traveled in an open field test.

40. The transgenic mouse of claim 36, wherein the transgenic mouse exhibits decreased susceptibility to seizure relative to a wild-type mouse.

41. The transgenic mouse of claim 40, wherein the decreased seizure susceptibility is characterized by an increased metrazol response threshold.

42. A method of identifying an agent that modulates a phenotype associated with the transgenic mouse of claim 36, the method comprising:

(a) administering an agent to the transgenic mouse; and

(b) determining whether the agent modulates the phenotype.

43. An agent identified by the method of claim 42.

44. An agonist or antagonist of CRFR2.

45. A cell derived from the transgenic mouse of claim 36.

46. Phenotypic data associated with the transgenic mouse of claim 36, wherein the data is in a database.

47. A transgenic mouse comprising a homozygous disruption in an NPY6-R gene.

48. The transgenic mouse of claim 47, wherein the transgenic mouse exhibits increased agility or coordination relative to a wild-type mouse.

49. The transgenic mouse of claim 48, wherein the increased agility or coordination is characterized by increased latency in an accelerating rotarod test.

50. A method of identifying an agent that modulates a phenotype associated with the transgenic mouse of claim 47, the method comprising: (a) administering an agent to the transgenic mouse; and

(b) determining whether the agent modulates the phenotype.

51. An agent identified by the method of claim 50.

52. An agonist or antagonist of NPY6-R.

53. A cell derived from the transgenic mouse of claim 47.

54. Phenotypic data associated with the transgenic mouse of claim 47, wherein the data is in a database.

55. A transgenic mouse comprising a homozygous disruption in an mGluR8 gene.

56. The transgenic mouse of claim 55, wherein the transgenic mouse exhibits increased activity relative to a wild-type mouse.

57. The transgenic mouse of claim 56, wherein the transgenic mouse is hyperactive.

58. The transgenic mouse of claim 56, wherein the increased activity is characterized by increased distance traveled in an open field test.

59. The transgenic mouse of claim 55, wherein the transgenic mouse exhibits a stimulus-processing deficit relative to a wild-type mouse.

60. The transgenic mouse of claim 59, wherein the stimulus-processing deficit is characterized by decreased pre-pulse inhibition.

61. The transgenic mouse of claim 55, wherein the transgenic mouse exhibits a neuropsychological disorder.

62. The transgenic mouse of claim 55, wherein the transgenic mouse exhibits schizophrenic behavior.

63. A method of identifying an agent that modulates a phenotype associated with the transgenic mouse of claim 55, the method comprising:

(a) administering an agent to the transgenic mouse; and

(b) determining whether the agent modulates the phenotype.

64. An agent identified by the method of claim 63.

65. An agonist or antagonist of mGluR8.

66. A cell derived from the transgenic mouse of claim 55.

67. Phenotypic data associated with the transgenic mouse of claim 55, wherein the data is in a database.

68. A transgenic mouse comprising a homozygous disruption in a MAS receptor gene.

69. The transgenic mouse of claim 68, wherein the transgenic mouse exhibits increased anxiety relative to a wild-type mouse.

70. The transgenic mouse of claim 69, wherein the increased anxiety is characterized by increased expulsion of fecal boli.

71. The transgenic mouse of claim 69, wherein the increased anxiety is characterized by decreased percentage of time spent in the central region of an open field test.

72. The transgenic mouse of claim 68, wherein the transgenic mouse exhibits decreased susceptibility to seizure relative to a wild-type mouse.

73. The transgenic mouse of claim 72, wherein the decreased seizure susceptibility is characterized by an increased metrazol response threshold.

74. The transgenic mouse of claim 68, wherein the transgenic mouse exhibits decreased activity relative to a wild-type mouse.

75. The transgenic mouse of claim 74, wherein the transgenic mouse is hypoactive.

76. The transgenic mouse of claim 74, wherein the decreased activity is characterized by decreased distance traveled in an open field test.

77. A method of identifying an agent that modulates a phenotype associated with the transgenic mouse of claim 68, the method comprising:

(a) administering an agent to the transgenic mouse; and

(b) determining whether the agent modulates the phenotype.

78. An agent identified by the method of claim 77.

79. An agonist or antagonist of a MAS receptor.

80. A cell derived from the transgenic mouse of claim 68.

81. Phenotypic data associated with the transgenic mouse of claim 68, wherein the data is in a database.