The invention encompasses antagonists and agonists of IGPcRl 7, as well as compounds or nucleotide constructs that inhibit expression of the IGPcRl 7 gene (anti-sense and ribozyme molecules), or promote expression of IGPcRl 7 (wherein IGPcRl 7 coding sequences are operatively associated with promoters, enhancers, etc.). Highly preferred are the IGPcRl 7 protein products (especially soluble derivatives of IGPcRl 7, or truncated polypeptides lacking the TM or CD domains) and fusion protein products, antibodies and anti-idiotypic antibodies, antagonists or agonists (including compounds that modulate signal transduction which may act on downstream targets in the IGPcRl 7 signal transduction pathway) that can be used for therapy of such diseases, by inhibiting receptor activity.

Nucleotide constructs encoding functional forms of IGPcRl 7 and mutant forms of IGPcRl 7 are preferred embodiments of the invention, as their uses include employment in the genetic engineering of host cells. Other preferred embodiments of the invention are anti-sense and ribozyme molecules, preferred for use in "gene therapy" approaches in the treatment of disorders or diseases arising from the aberrant or altered activity of IGPcRl 7. The gene therapy vector alone or when incorporated into recombinant cells, may be administered in a suitable formulation for intravenous, intra-muscular, intra-peritoneal delivery, or may be incorporated into a timed release delivery matrix.

Transgenic and knock-out animal models

The animal-based and cell-based models can be used to identify drugs, biologicals, therapies and interventions which can be effective in treating disorders with aberrant expression or activity. IGPcRl 7 sequences can be introduced into, and over- expressed and/or can be disrupted in order to under-express or inactivate IGPcRl 7 gene expression.

In one embodiment of the invention, the IGPcRl gene products can also be expressed in transgenic animals. Non-human animals of any species, including, but not limited to, mice, rats, rabbits, guinea pigs, sheep, cows, goats, may be used to generate IGPcRl 7 transgenic animals. The present invention provides for transgenic animals that carry the IGPcRl 7 transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals. The transgene may be expressed in all tissues of the animal, or may be limited to specific tissues. Any technique known in the art may be used to introduce the IGPcRl 7 transgene into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to pronuclear microinjection (Hoppe PC and Wagner TE, U.S. Patent No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten et al, 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); gene targeting in embryonic stem cells (Thompson et al, 1989, Cell 56:313-321); electroporation of embryos (Lo, 1983, Mol. Cell. Biol., 3:1803-1814); and sperm- mediated gene transfer (Lavitrano et al, 1989, Cell 57:717-723); etc. For a review of such techniques, see Gordon, 1989, "Transgenic Animals", Intl. Rev. Cytol.

115:171-229, which is incorporated by reference herein in its entirety.

The present invention relates to knock-out animals engineered by homologous recombination to be deficient in the production of the IGPcRl 7. The present invention is directed to a knock-out animal having a phenotype characterized by the substantial absence of IGPcRl 7, otherwise naturally occurring in the animal. In addition, the invention encompasses the DNA constructs and embryonic stem cells used to develop the knock-out animals and assays which utilize either the animals or tissues derived from the animals. Preferably, these cells, tissues and cell lines are characterized by the substantial absence of IGPcRl 7 that would otherwise be naturally occurring in their normal counterparts. Gene targeting is a procedure in which foreign DNA sequences are introduced into a specific locus within the genome of a host cell. In another embodiment of the invention, endogenous IGPcRl 7 gene expression can be reduced by inactivating or knocking out the IGPcRl 7 gene or its promoter using targeted homologous recombination, (e.g., see Smithies et al, 1985, Nature 317:230-234; Thomas & Capecchi, 1987, Cell 51 :503-512; Thompson et al, 1989, Cell 5:313-321 ; each of which is incorporated by reference herein in its entirety). For example, a mutant, non-functional IGPcRl 7 (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous IGPcRl 7 gene (either the coding regions or regulatory regions of the IGPcRl 7 gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express IGPcRl 7 in vivo. Insertion of the DNA construct, via targeted homologous recombination into the genome, results in abolishing IGPcRl 7 gene function.

One preferred technique for targeted mutagenesis in this invention is based on homologous recombination. The general methodologies of targeting mutations into the genome of cells, and the process of generating mouse lines from genetically altered embryonic stem (ES) cells with specific genetic lesions are well known (Bradley, 1991, Cur. Opin. Biotech. 2: 823-829). See also U.S. patents 5,557,032 by

Mak et al, and U.S. Patent No. 5,487,992 by Capecchi et al , included by reference herein. Preferred in this invention is a synthetic recombination vector which contains the genetic information of the targeted chromosomal locus recombines with the genomic DNA after introduction into a cell. A strategy of "positive/negative selection" can be used to enrich the cell population for cells in which targeting vectors have integrated into the host cell genome, and recombination has occurred at the desired gene locus (Mansour, et al, 1988, Nature 336:348). The vector usually contains a positive selection cassette which is flanked by the genetic information of the target locus to enrich for cells where the vector successfully recombines with the chromosomal DNA against the pool of non-recombinant cells. The likelihood of obtaining an homologous recombination event increases with the size of the chromosomal vector DNA and is further dependent on the isogenicity between the genomic DNA of the vector and the target cell (See te Reile et al, 1992, P.N.A.S. USA 89:5128-5132; Deng et al, 1991, Mol. Cell. Biol., 12, 3365-3371). Also preferred in this invention are large stretches of genomic DNA flanking the

IGPcRl 7 gene ortholog in the target animal species. The cloning of large chromosomal fragments of the target gene, the sub-cloning of this DNA into a bacterial plasmid vector, the mapping of the gene structure, the integration of the positive selection cassette into the vector and finally, the flanking of one or both homologous vector arms by a negative selection marker are well described in the literature. Also preferred are replacement-type targeting vectors using yeast host cells are described by Storck et al, 1996, Nuc. Acids Res. 24:4594-4596. The use of other vectors such as bacteriophage λ and vectors for phage-plasmid recombination have been described by Tsuzuki et al, 1998, Nuc. Acids Res 26:988-993; transposon-generated gene targeting constructs have also been described by

Westphal et al, 1997, Curr. Biol., 7:530-533 and are within the scope of the invention.

The most highly preferred method in this invention is described by Wattler S & Nehls M, German patent application DE 100 16 523.0, "Klonierungssystem zur

Konstruktion von homologen Rekombiationsvektoren", filed April 03, 2000, included by reference in whole herein,, and described in part in Example 7. This method reduces the time required for the construction of such vectors from 3-6 months to about 14 days. The vector includes a linear lambda vector (lambda-KO- St?) that comprises a stuffer fragment; an E. coli origin of replication; an antibiotic resistance gene for bacterial selection, two negative selection markers suitable for use in mammalian cells; LoxP sequences for cre-recombinase mediated conversion of linear Lambda phages into high copy plasmids. In a final targeting vector, the stuffer fragment is replaced by nucleotide sequences representing a left arm of homology, an ES cell selection cassette, and a right arm of homology. The transformation of mouse 129 ES cells with the final vector construct is done according to standard procedures. The targeting vector is linearized and then introduced by electroporation into ES cells. Cell clones are positively selected with G418 and negatively selected with GANC (ganciclovir, 0.2 μM). Targeted ES-cell clones with single integration sites are identified, confirmed by hybridization, and expanded in culture for injection.

The invention also encompasses embryonic stem (ES) cells derived from a developing mouse embryo at the blastocyst stage, that are modified by homologous recombination to contain a mutant IGPcRl 7 gene allele. The modified ES cells are reintroduced into a blastocyst by microinjection, where they contribute to the formation of all tissues of the resultant chimeric animal, including the germ line (Capecchi, 1989, Trends Genet., 5:70; Bradley, et al, 1984, Nature, 309:255). Modified ES cells may also be stored before reimplantation into blastocysts. The chimeric blastocysts are implanted into the uterus of a pseudopregnant animal, prepared by mating females with vasectomized males of the same species. Typically chimeras have genes coding for a coat color or another phenotypic marker that is different from the corresponding marker encoded by the stem cell genes.

Also within the scope of the invention are chimeric male non-human animals and their heterozygous offspring carrying the IGPcRl 7 gene mutation which are bred to obtain progeny which are homozygous for the mutation, preferred animals being mice. A phenotype selection strategy may be employed, or chromosomal DNA may be obtained from the tissue of offspring, screened using Southern blots and/or PCR amplification for the presence of a modified nucleotide sequence at the IGPcRl 7 gene locus, liked described in the above section of identifying positivlly targeted ES cells. Other means for identifying and characterizing transgenic knock-out animals are also available. For example, Northern blots can be used to probe mRNA obtained from tissues of offspring animals for the presence or absence of transcripts coding for either the IGPcRl 7, the marker gene, or both. In addition, Western blots might be used to assess IGPcRl 7 expression by probing with antibody specific for the receptor.

These animals are characterized by including, but not limited to, a loss in the ability to bind ligands specific for IGPcRl 7 and/or by a loss in expression from the IGPcRl 7 gene locus. Preferably, the animals produce no functional forms of

IGPcRl 7 at all. Once homozygous transgenic animals have been identified, they may preferably be interbred to provide a continual supply of animals that can be used in identifying pathologies dependent upon the absence of a functional IGPcRl 7 and in evaluating drugs in the assays described above. Also highly preferred in this invention, are these animals as providing a source of cells, tissues and cell lines that differ from the corresponding cells, tissues and cell lines from normal animals by the absence of fully functional forms of IGPcRl 7.

The methodology needed to make such animals can be adapted to any non-human animal, preferably rodents such as hamsters, rats or mice, and most preferably, mice.

In another embodiment, clones of the non-human transgenic animals can be produced according to methods described in Wilmut et al, 1997, Nature, 385:810- 813.

EXAMPLES

Example 1. Identification of a full-length human cDNA coding for a novel GPcR, IGPcR17.

A coding sequence of 1026 bp was identified as follows from the EMBL alert HTGH (High Throughput Genome) database (see Fig.l, SEQ ID NOT). A search was performed using the nucleotide sequences of known GPcRs. A sequence with a statistically significant score was returned and searched for open reading frames. Subsequently a putative coding region was assigned and used in primer design. The tracked human genomic IGPcRl 7 sequence contains the full-length cDNA sequence, identified as a single exon coding GPcR. IGPcRl 7 encodes a protein of 343 amino acids, (see Fig.2, SEQ ID NO:2). A BLASTP search (Basic Local Alignment Search Tool for Proteins, National Institutes of Health, Bethesda MD, U.S.A.) revealed that the human protein most closely related to human IGPcRl 7 is KIAAOOOl, a human G protein-coupled receptor of 338 amino acids (Genpept. accession number NP055694; Nomura et al, 1994, DNA Res. 1 :27-35; Nomura et al, 1994, DNA Res. 1 :47-56). IGPcR17 has 47% identity and 66%> sequence homology with conserved substitutions to KIAAOOOl.

Example 2. Tissue-specific expression of human IGPcRlJ, analysis by RT- PCR.

A panel of cDNAs derived from total RNA from 29 human tissues (Clontech Laboratories, Inc., Palo Alto CA, USA; Invitrogen Corp., Carlsbad CA, USA) was tested in a reverse transcription-polymerase chain reaction (RT-PCR) assay. The sequence of the primers used to amplify a 466 bp product (SEQ ID No: 5), which spans a region including transmembrane domains 1 to 4, is as follows: 5'- CAC TGC TCT ACA CTG TCC TG (coding sequence position 86 to 105; SEQ ID NO: 3) 5'- GAC CGA ACT CTG ATT TAA GG (coding sequence position 501 to 486;

SEQ ID NO:4)

The conditions for the PCR were: denaturation at 94°C for 45 seconds, annealing at 56°C for 1 minute, and extension at 72°C for 30 seconds, for a total of 35 cycles, in a Thermocycler (MJ Research, Watertown MA, USA; type PTC-225). The PCR products were analyzed on an 1.8% agarose gel and stained with ethidium bromide to visualize DNA by ultraviolet imaging. The tissues analyzed were: skin, whole brain, fetal brain, cerebellum, thymus, esophagus, trachea, lung, breast, mammary gland, heart, liver, fetal liver, kidney, spleen, adrenal gland, pancreas, stomach, small intestine, skeletal muscle, adipose tissue, uterus, placenta, bladder, prostate, testis, colon, rectum and cervix. Positive (human genomic DNA) and negative (water) controls were included.

Positive PCR products of 466 base pairs in size were observed in the human panel, in order of decreasing signal intensity, in whole brain, fetal brain, cerebellum, placenta, skin, adipose tissue, uterus, pancreas, spleen, fetal liver and trachea. The correct identity of the sequence amplified was confirmed by sequencing of the PCR products.

Example 3. Tissue-specific expression of human IGPcR17 transcript, analysis by Northern hybridization.

Northern hybridization of polyA+ RNAs from several human tissues was carried out using a IGPcRl 7 specific DNA-probe. The probe was generated by radiolabelling the purified and sequenced PCR product generated using primers as described in

Example 2. The probe spans sequences coding for transmembrane regions 1 to 4 and is 466 bp in length. Commercially available Multiple Tissue Northern Blots (Clontech Laboratories, Palo Alto CA, USA) containing approximately 2 micrograms of poly A + RNA per lane, adjusted to provide a consistent beta-actin signal in each lane, were hybridized, following the manufacturer's instructions.

These blots are optimized to give best resolution in the 1.0-4.0 kb range, and marker RNAs of 9.5, 7.5, 4.4, 2.4, 1.35 and 0.24 kb are run for reference. In addition, a different set of commercially available Multiple Tissue Northern Blots (BioChain Institute, Hayward CA, USA) containing 3 micrograms of human tissue poly A + RNA per lane, was hybridized. Membranes were pre-hybridized for 30 minutes and hybridized overnight at 68°C in ExpressHyb hybridization solution (Clontech Laboratories, Palo Alto CA, USA) as per the manufacturer's instructions. The cDNA probe used was labeled with [α P] dCTP using a random primer labeling kit (Megaprime DNA labeling system, Amersham Pharmacia Biotech, Piscataway NJ, USA) and had a specific activity of 1 x 10⁹ dpm/μg. The blots were washed several times in 2X SSC, 0.05% SDS for 30-40 min at room temperature, and were then washed in 0.1X SSC, 0.1% SDS for 40 min at 50°C (see Sambrook et al, 1989, "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Press, New York, USA). The blots were covered with standard domestic plastic wrap and exposed to X-ray film at -70°C with two intensifying screens for 36 hours.

The tissues represented in the Clontech Laboratories Multiple Tissue Northern Blots are as follows:

Blot #1 Blot #2

Heart Stomach

Brain Thyroid

Placenta Spinal Cord

Lung Lymph Node

Liver Trachea

Skeletal Muscle Adrenal Gland

Kidney Bone Marrow

Pancreas

The results of this experiment indicate that IGPcRl 7 is expressed as an approximately 2.4 kb transcript in human brain & spinal cord (see Fig. 3a). A less intense signal was obtained at a lower molecular weight of about 1.8 kb in the trachea. This band is most probably a cross-reactive RNA species, as it is also observed upon hybridization with probes detecting a variety of GPcRs.

The tissues represented in the BioChain Institute Multiple Tissue Northern Blots are as follows:

Blot #1 Blot #11 Blot #V Blot#NIII

Heart Stomach Uterus Brain

Brain Jejunum Cervix Kidney

Liver Ileum Ovary Spleen Pancreas Colon Testis Intestine

Skeletal Muscle Lung Prostate Uterus

Lung Lung Cervix

Placenta

Lung

The results of this experiment indicate that IGPcRl 7 is expressed most strongly in a predominant 2.4 kb transcript in human brain. A fainter transcript species of about 4.4 kb in size is also detected in brain. Additional tissues expressing low levels of the IGPcR17 2.4 kb transcript are skeletal muscle, lung, intestine, placenta, uterus, cervix and prostate (see Fig. 3b).

Example 4. Tissue-specific expression and relative abundance of human IGPcRl 7 transcript, dot blot analysis.

Dot blot analysis of the IGPcRl 7 transcript in polyA+ RNAs from several human tissues was performed. An IGPcRl 7-specific DNA probe was generated by radiolabeling the purified and sequenced PCR product generated using primers as described in Example 2. The probe spans sequences coding for transmembrane domains 1 to 4 and is 466 bp in length. A commercially available Multiple Tissue Expression Array membrane (Clontech Laboratories, Inc., Palo Alto CA, USA, cat. no. 7775-1) containing polyA + RNA in each 1mm dot, normalized to the mRNA levels of eight housekeeping genes, was hybridized (see Fig. 4a and 4b). Thus changes seen can be attributed to actual differences in mRNA abundance. The membrane was pre-hybridized for 30 minutes and hybridized overnight, at 65°C in ExpressHyb Hybridization Solution as per the manufacturer's instructions (Clontech Laboratories, see above). The cDNA probe used was labeled with [α³²P] dCTP using a random primer labeling kit (Megaprime DNA labeling system, Amersham Pharmacia Biotech, Piscataway NJ, USA) and had a specific activity of 1 xlO⁹ dpm/μg. The blot was washed several times in 2X SSC, 0.05% sodium dodecyl sulfate (SDS) for 30-40 minutes at room temperature, then washed in 0.1X SSC, 0.1% SDS for 40 min at 50°C (see Sambrook et al, 1989, "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Press, New York, USA). The blot was covered with plastic wrap and an X-ray film exposure made for two days at -70°C using two intensifying screens.

The results of this experiment indicate that IGPcRl 7 is expressed in all regions of the human brain and CNS examined, such as whole brain, cerebellum, substantia nigra, cerebral cortex, accumbens nucleus, frontal lobe, corpus callosum, thalamus, parietal lobe, amygdala, occipital lobe, caudate nucleus, spinal cord, temporal lobe, paracentral gyrus of cerebral cortex, medulla oblongata, pons and putamen, as well as, at a lower level in fetal brain (see Figure 4a).

Example 5. Characterization of human IGPcRlJ protein.

Hydrophobic analysis of the predicted amino acid sequence showed seven hydrophobic regions corresponding to the seven transmembrane regions, a conserved structural feature of G protein-coupled receptors. Human IGPcRl 7 is a protein of 342 amino acid residues. The encoded protein was compared to amino acid sequences present in public databases EMBL and Genbank. IGPcRl 7 has 47% identity and 66% similarity to amino acid sequences of KIAAOOOl, a G protein- coupled receptor of 338 amino acids in size (Genpept accession number NP055694; Nomura et al, 1994, DNA Res., 1:27-35; Nomura et al, 1994, DNA Res., 1 :47-56). Recently a rat ortholog of human KIAAOOOl, NTR 15-20 (Genpept accesion number

0355881), encoding a protein of 305 amino acids was isolated and shown to have 81%) amino acid identity, and 92%) sequence homology (with conserved substitutions) to KIAAOOOl (Charlton et al, 1997, Brain Res., 764:141-148).

Fig. 7a shows the amino acid sequence of IGPcR17 ('query') compared to the amino acid sequence of the mouse ortholog of IGPcRl 7 ('sbjct'), and Fig. 7b shows the amino acid sequence of IGPcRl 7 ('query') compared to the amino acid sequence of human KIAAOOOl ('sbjct'); as abstracted from the SWISSPROT database and analyzed using a BLASTP alignment program. The predicted transmembrane domains of IGPcR17 are flanked by amino acids 29-50 (TM1), 61-80 (TM2), 100- 124 (TM3), 142-160 (TM4), 190-212 (TM5), 238-257 (TM6), 282-306 (TM7), as underlined. Human IGPcRl 7 protein and the mouse ortholog IGPcRl 7 protein exhibit an amino acid identity of 88.7% in an overlap of 337 residues.

Fig. 8 shows a hydropathy plot for the predicted amino acid sequence of the human IGPcRl 7 protein compared to the sequences predicted for human KIAAOOOl and the mouse ortholog IGPcRl 7. The analysis was performed using the method of Kyte and DooLittle (1982, J. Mol. Biol., 157:105-32), with the DAMBE program (Data Analysis in Molecular Biology and Evolution), University of Hong Kong, version 4.0.41.

Example 6. Identification of mouse ortholog of human IGPcRl 7.

The mouse ortholog gene to the human IGPcRl 7 gene was identified by a BLAST search of public databases EMBL and Genbank using the full-length coding sequence of the human gene as a query sequence. The highest score was assigned to an EMBL EST/MM 1179620, Soares mouse 3NbMs Mus musculus cDNA clone

IMAGE 764455; length 542 bp. The open reading frame was used in primer design.

From mouse strain 129, genomic clones containing the full-length cDNA (SEQ ID NO:6, Fig. 5) and flanking genomic sequences were isolated and were utilized in the construction of targeting vectors. The cDNAs of the human and mouse IGPcRl 7 were found to be 90 %> identical. Several clones were isolated from a mouse 129 genomic library by hybridization of a probe corresponding to a 356 bp PCR product, as described in Example 7. Example 7. Tissue-specific expression of mouse orthologue gene, analysis by RT-PCR.

A panel of cDNAs derived from total RNA isolated from 22 mouse tissues was tested in a reverse transcription-polymerase chain reaction (RT-PCR) assay. The sequence of the primers used to amplify a 356 bp product (SEQ ID NO: 10), which includes predicted transmembrane domains 3 to 5, is as follows: 5'- CGT TCC TGG GGT TGA TAA CC (coding sequence position 355- 374; SEQ ID NO:8) 5'- CTT GGG AAC TTT GGC TGA AC (coding sequence position 710-691 ; SEQ

ID NO:9)

The conditions for the polymerase chain reaction (PCR) were: denaturation at 94°C for 45 seconds, annealing at 56°C for 1 minute and extension at 72°C for 30 seconds, for a total of 35 cycles, in a Thermocycler (MJ Research; type PTC-225). The PCR products were analyzed on an 1.8% agarose gel and stained with ethidium bromide to visualize DNA by ultraviolet imaging. The tissues analyzed were: brain, brain without cerebellum, cerebellum, thymus, eye, tongue, trachea, adipose tissue, lung, heart, spleen, kidney, liver, stomach, small intestine, large intestine, colon, rectum, bladder, uterus, prostate, testis. Positive (mouse genomic DNA) and negative (water) controls were included.

Positive PCR products of 356 bp in size were observed in the mouse panel in order of decreasing signal intensity in whole brain, brain without cerebellum, cerebellum, uterus, trachea, eye, bladder and rectum.

Example 8. Tissue-specific expression of mouse IGPcR17, analysis by Northern hybridization.

RNA originating from several tissues was isolated, separated by agarose gel electrophoresis and blotted onto a Hybond N+ Nylon membrane (Amersham Pharmacia Biotech, Piscataway NJ, USA). After covalent fixation to the membrane by UV crosslinking, the Northern blot was hybridized with a mouse IGPcR-17 probe, as indicated in Example 7. Tissues analyzed are: total brain, cerebrum, cerebellum, heart, spleen, testis, epididymis and uterus.

A single transcript of about 2.4kb in size, was detected, being strongly expressed in total brain, cerebrum and cerebellum, and expressed in testis and epididymis to a weaker extent. In heart and spleen a single faint transcript species of about 2.8 kb in size was detected, as depicted in Figure 9.

Example 9. Characterization of mouse IGPcR17 protein.

Hydrophobic analysis of the predicted amino acid sequence of the mouse ortholog protein of IGPcRl 7 indicated seven hydrophobic domains corresponding to the seven transmembrane regions, a conserved structural feature of G protein-coupled receptors. The mouse IGPcRl 7 protein (Fig.6, SEQ ID NO:7) is a protein of 347 amino acid residues. The encoded protein has 88.7% identity to the amino acid sequence of the human IGPcRl 7 protein.

Figure 7a shows the amino acid sequence of human IGPcRl 7 ('query') compared to that of the mouse ortholog ('sbjct'), as abstracted from the SWISSPROT database and analyzed using the BLASTP alignment program. The predicted transmembrane domains (TM 1 to 7) of mouse IGPcR17 are flanked by amino acids 34-55 (TM1),

69-86 (TM2), 99-121 (TM3), 144-167 (TM4), 192-214 (TM5), 240-262 (TM6), 279-301 (TM7), as underlined.

Figure 8 shows a hydropathy plot for the predicted sequence of the mouse IGPcRl 7 protein compared to the human IGPcRl 7 protein and that of human KIAAOOOl . The analysis was performed using the method of Kyte and DooLittle (1982, J. Mol. Biol, 157:105-32), with the DAMBE program (Data Analysis in Molecular Biology and Evolution), University of Hong Kong, version 3J.49.

Example 10. Generation of ES cells with a modified IGPcR17 allele, produced by homologous recombination.

The most preferred method in this invention is described in Wattler S & Nehls M, German patent application DE 100 16 523.0, "Klonierungssystem zur Konstruktion von homologen Rekombiationsvektoren", filed April 03, 2000. This method reduces the time required for the construction of such a vector from 3-6 months to about 14 days. The vector includes a linear lambda vector (lambda-KO-Sfi) that comprises a stuffer fragment; an E. coli origin of replication; an antibiotic resistance gene for bacteria selection, two negative selection markers suitable for use in mammalian cells; LoxP sequences for cre-recombinase mediated conversion of linear lambda phages into high copy plasmids. In a final targeting vector, the stuffer fragment is replaced by nucleotide sequences representing a left arm of homology, an ES cell selection cassette, and a right arm of homology.

To abolish the gene function of mouse IGPcRl 7 (mIGPcR17) a deletion of approximately 880 bp of the coding region starting approximately 10 bp upstream of the ATG was performed (see Fig. 10). The left arm of homology (hereafter referred to as A/C) is PCR amplified with the primers C and A. The primers contain Sfi I restriction sites A and C in their 5 '-ends, respectively. Sfi recognizes and cuts the nucleotide sequence 5-GGCCNNNNNGGCC-3'. By changing the nucleotides designated N, unique and non-compatible Sfi restriction sites are generated. The 3'- end of primer A is homologous to 25 bp of mouse IGPcRl 7, ending with the 10 bp downstream of the ATG. The 3 '-end (25 bp) of primer C is homologous to a position approximately 2500 basepairs upstream of the ATG. The right arm of homology (hereafter referred to as B/D) is PCR amplified with primers B and D: B is located approximately 880 bp downstream of the ATG, and D approximately 2000 bp downstream of the stop codon. Both primers contain S^z-restriction sites B or D in their 5'-ends, respectively. To avoid the introduction of point mutations the Expand high fidelity PCR-System, (Boehringer Mannheim / Roche Diagnostics, Basel CH) is used. A ligation of A/C with B/D and a selection cassette leads to an approximately 880 bp deletion of the mIGPcR17 coding region, thereby creating a null allelle. Both PCR-products A/C and B/D are purified using Qiaquick PCR Purification Kit according to the manufacturer (Quiagen, Nenlo, ΝL). The PCR-products are cleaved

3 hours at 50°C with 60 U Sfi and subsequently purified (Qiaquick PCR Purification kit). The final volume is 30 μl/product. The ES-cell selection cassette (IRES-β- lactamase-MCSneo) contains Sfi-sites A and B 5'- and 3'-, respectively (Wattler S, et al, 1999, Biotechniques, 26:1150-1159). A typical ligation is 50 ng lambda-KO-Sfi- arm (S z-cleaved), 10 ng selection cassette, 1 ng A/C, 1 ng B/D, 1 x ligation buffer and 1U T4 ligase (Boehringer Mannheim / Roche Diagnostics, Basel CH). The ligation is carried out for 2 hours at room temperature. Two μl of the ligation are used for in vitro packaging ('Gigapack plus' from Stratagene, La Jolla CA, USA) for 1.5 hours at room temperature according to the manufacturer's instructions. Aliquots of 10 μl and 50 μl are used to infect C600 bacteria (Stratagene, La Jolla CA, USA) and infection is performed overnight. Single plaques in SM-buffer (Ausubel FM et al, 1994, "Current Protocols in Molecular Biology", John Wiley & Sons, New York) are taken to infect BNN 132 bacteria (30 min at 30°C) for plasmid conversion and infection. Bacteria are cultured over 16 hours at 30°C in TB media (Ausubel FM et al, 1994, "Current Protocols in Molecular Biology", John Wiley & Sons, New

York), containing 100 μg/ml ampicillin (Amersham Pharmacia Biotech, Piscataway NJ,JJSA; cat. no. US 11259-25). Plasmids are harvested using the Qiagen plasmid kit (Qiagen cat. no. 12143) according to the manufacturer's instructions. To verify plasmid integrity, Sfi and EeoR -digests are performed.

The transformation of mouse 129 ΕS cells with the final targeting vector is performed according to standard procedures. Electroporated 129 mouse ES cells are double-selected with G418 (400 μg/ml) for 7 days and GANC (ganciclovir, 0.2 μM) for 3 days, starting on day 3 after electroporation, for positive and negative selection, respectively, thereby enriching for transformants having the neomycin resistance gene integrated into an endogenous IGPcRl 7 allele. Single cell clones are propagated, frozen down and expanded for DNA isolation. To identify positively targeted clones, ES cell DNA is isolated from selected clones, incubated with an appropriate restriction enzyme, and the digestion products separated on an agarose gel. Southern blots are hybridized with a 5' external probe and positive targeted candidates are verified by hybridization with a 3' external probe. A single integration is confirmed by hybridization with a probe derived from the neomycin gene. Positive ES cells are isolated and expanded in culture.

Example 11. Mice Deficient in the Expression of the IGPcR17 Gene.

Male chimeric mice are generated by micro-injection of ES cells carrying a recombined allele into 129/SvEv mouse blastocysts, using standard methodology. The chimeric blastocyst is implanted into the uterus of a pseudopregnant mouse, prepared by mating females with vasectomized males of the same species. The chimeras are bred to wild type animals. Tail DNA is isolated from the offspring of these chimeric mice and analyzed by incubation with appropriate restriction enzymes followed by Southern analysis, using the same strategy as outlined above to determine germline transmission. The blots demonstrate the transmission into the mouse genome of the mutation altering the IGPcRl 7 allele in transformant ES cells.

The chimeric male mouse and its heterozygous progeny (+/-) are bred to produce mice homozygous for the mutation (-/-).

Northern blots are used to probe mRNA obtained from tissues of offspring for the presence or absence of transcripts encoding either the IGPcR27, the marker gene, or both. In addition, Western blots are used to assess IGPcR27 expression by probing with antibody specific for the receptor.

Those skilled in the art will be able to recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the invention.

Claims

1. An isolated nucleic acid molecule, wherein said nucleic acid molecule comprises at least one of: (a) the nucleotide sequence of SEQ ID NO : 1 ;

(b) the nucleotide sequence of SEQ ID NO:6;

(c) a nucleotide sequence which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or any unique fragment thereof wherein the amino acid sequence of the fragment is greater than ten amino acids in length.

(d) a nucleotide sequence which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:7, or any unique fragment thereof wherein the amino acid sequence of the fragment is greater than ten amino acids in length.

2. An isolated nucleic acid molecule comprising an allelic variant of a nucleotide sequence which encodes a polypeptide comprising the amino acid sequence selected from the group of:

(a) the amino acid sequence of SEQ ID NO:2; and (b) the amino acid sequence of SEQ ID NO:7; wherein said allelic variant contains at least 80% nucleic acid homology and hybridizes to the complement of SEQ ID NO:l under highly stringent conditions equivalent to hybridization in 42°C in a hybridization solution comprising 50% formamide, 1% SDS, 1M NaCl, 10% Dextran sulfate, and washing twice for 30 minutes in a wash solution comprising 0. lxSSC and 1% SDS.

3. The isolated nucleic acid molecule of claims 1 or 2, comprising a nucleotide sequence which encodes at least one of the group of polypeptides, peptides and fusion proteins, comprising an amino acid sequence at least 70% similar to an amino acid sequence selected from the group of:

(a) the amino acid sequence of SEQ ID NO:2; and (b) the amino acid sequence of SEQ ID NO:7.

4. The isolated nucleic acid molecule of claims 1 to 3 operatively linked with a nucleotide regulatory sequence capable of controlling expression of the nucleic acid molecule in a host cell or non-human animal. '"

5. A vector comprising the isolated nucleic acid molecule of any of claims 1 to 4.

6. A host cell genetically engineered to contain at least one of: (a) the nucleic acid molecule of any of claims 1 to 4; or

(b) the vector of claim 5.

7. The host cell of claim 6 wherein said host cell is a eucaryotic cell, being at least one of: (a) a yeast cell;

(b) an insect cell; or

(c) a mammalian cell.

8. The human IGPcRl 7 protein of SEQ ID NO:2, or any unique fragment thereof wherein the amino acid sequence of the fragment is greater than ten amino acids in length, including but not limited to polypeptides, peptides, isolated domains and fusion proteins.

9. The mouse IGPcRl 7 protein of SEQ ID NO:7, or any unique fragment thereof wherein the amino acid sequence of the fragment is greater than ten amino acids in length, including but not limited to polypeptides, peptides, isolated domains and fusion proteins.

10. Antibodies specifically targeting the IGPcRl 7 proteins of any of claims 8 or 9, and/or polypeptides, peptides, isolated domains and the the IGPcRl 7 component of fusion proteins of said IGPcRl 7 proteins.

11. Agonists and antagonists of IGPcRl 7 protein that compete selectively with native natural IGPcRl 7 ligand and which modulate IGPcRl 7 gene expression or gene product activity, including: (a) 'small molecules' of molecular mass less than 6 kDa; (b) molecules of intermediate size, having molecular mass between 5 kDa to 15 kDa; and (c) large molecules of molecular mass greater than 12 kDa; the latter including mutant natural IGPcRl 7 ligand proteins that compete with native natural IGPcRl 7 ligand and which modulate IGPcRl 7 gene expression or gene product activity.

12. Anti-sense and ribozyme molecules that can be used to inhibit IGPcRl 7 gene expression or expression constructs used to enhance IGPcRl 7 gene expression.

13. Methods of identifying compounds of any of claims 11 or 12, which modulate the activity of IGPcRl 7 or IGPcRl 7 gene expression.

14. Embryonic stem cells containing a disrupted endogenous IGPcRl 7 gene.

15. Non-human knock-out animals that do not express IGPcRl 7, wherein the endogenous animal ortholog of the IGPcRl 7 gene is functionally disrupted.

16. The non-human knock-out animals of claim 15, wherein the endogenous animal ortholog of the IGPcRl 7 gene is functionally disrupted by an homologous recombination method.

17. Mutated non-human animals that express a non-functional or partially functional form of IGPcRl J

18. A non-human transgenic animal model expressing the human IGPcRl 7 cDNA sequence as shown in SEQ ID NO:l or the nucleic acid molecule of any of claims 1 to 4.

19. The non-human animal model according to any one of claims 17 to 18, whereby the human IGPcRl 7 is encoded by a nucleic acid sequence which is homozygous in said animal model.

20. Progeny of non-human animals of any of claims 15 to 19, including both heterozygous and homozygous offspring.

21. Non-human animals of any of claims 15 to 20, wherein the animal is from a genus selected from the group consisting of Mus (e.g., mice), Rattus (e.g., rats),

Oryctologus (e.g., rabbits) and Mesocricetus (e.g., hamsters).

22. Use of the non-human animal according to any one of claims 15 to 21, for the dissection of the molecular mechanisms of the IGPcRl 7 pathway, for the identification and cloning of genes able to modify, reduce or inhibit the phenotype associated with IGPcRl 7 activity or deficiency.

23. Use of the animal model according to any of claims 15 to 21 for the identification of gene and protein diagnostic markers for diseases.

24. Use of the animal model according to any of claims 15 to 21 for the identification and testing of compounds useful in the prevention, amelioration or treatment of diseases associated with IGPcRl 7 activity or deficiency.

25. The use of any of claims 23 or 24 wherein the disease is selected from the group of psychiatric and central nervous system diseases associated with signal processing in the central nervous system.

26. The use of claims 25 wherein the disease is selected from the group of learning and memory disorders, movement dysfunctions, tics, tremor, Tourette's syndrome,

Parkinson's disease, Huntington's disease, dyskinesias, dystonia, pain and spasms.

27. A method of identifying compounds suitable for modulating the activity of the protein according to claim 8, for treatment of diseases characterized by aberrant expression or activity of IGPcRl 7.

28. A method of prevention, amelioration or treatment of diseases characterized by aberrant expression or activity of IGPcRl 7, by the administration of compounds that bind specifically to the IGPcRl 7 gene or protein and/or which modulate IGPcRl 7 expression or IGPcRl 7 activity; the compounds that that bind specifically to the IGPcRl 7 gene or protein and/or which modulate IGPcRl 7 expression or IGPcRl 7 activity for the prevention, amelioration or treatment of diseases characterized by aberrant expression or activity of IGPcRl 7; and the use of compounds that that bind specifically to the IGPcRl 7 gene or protein and/or which modulate IGPcRl 7 expression or IGPcRl 7 activity for prevention, amelioration or treatment of diseases characterized by aberrant expression or activity of IGPcRl 7.

29. A gene therapy method of prevention, amelioration or treatment of diseases characterized by aberrant expression or activity of IGPcRl 7, by the administration of vectors and/or host cells containing nucleotide sequences according to any of claims 1 to 7, that modulate IGPcRl 7 expression or IGPcRl 7 activity; the vectors and/or host cells containing nucleotide sequences according to any of claims 1 to 7 which modulate IGPcRl 7 expression or IGPcRl 7 activity for the prevention, amelioration or treatment of diseases characterized by aberrant expression or activity of IGPcRl 7; and the use of vectors and/or host cells containing nucleotide sequences according to any of claims 1 to 7 which modulate IGPcRl 7 expression or IGPcRl 7 activity for prevention, amelioration or treatment of diseases characterized by aberrant expression or activity of IGPcRl 7.

30. The method of any of claims 27 to 29 wherein the disease is selected from the group of psychiatric and central nervous system diseases associated with signal processing in the central nervous system.

1. The method of claim 30 wherein the disease is selected from the group of learning and memory disorders, movement dysfunctions, tics, tremor, Tourette's syndrome, Parkinson's disease, Huntington's disease, dyskinesias, dystonia, pain and spasms.