Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
2001-07-06
2004-07-20
Falk, Anne-Marie (Department: 1636)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S003000, C800S025000
Reexamination Certificate
active
06765126
ABSTRACT:
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
Many medically significant biological processes are mediated by proteins participating in signal transduction pathways that involve G-proteins and/or second messengers such as cAMP. The membrane protein gene superfamily of G-protein coupled receptors (GPCRs) include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors. 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 &agr;-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 that 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 &agr;-subunits preferentially stimulate particular effectors to modulate various biological functions in a cell.
One important subfamily of the GPCRs is the neuropeptide Y receptor (NPYR) 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. NYAcad. 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 exposures 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
14, 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 (
J. Pharmacol. Exp. Ther.
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 Y1, 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 cds 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 sequence (GenBank GI or NID number: 1378003; Accession number: U58367) comprises 2281 bp; the coding region is believed to comprise bases 823-1938.
More recently, a transgenic mouse with a disrupted NPY-Y1 gene has been reported (U.S. Pat. No. 5,817,912); this “knockout” mouse was studied with respect to eating behavior and weight changes.
Given the importance of GPCRs, particularly the neuropeptide receptors, a clear need exists for identification and characterization of GPCRs which can play a role in preventing, ameliorating or correcting dysfunctions or diseases.
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 NPY6 receptor (NPY6-R) gene. The transgenic cells of the present invention are comprised of any cells capable of undergoing homologous recombination. Preferably, the cells of the
Deltagen Inc.
Driscoll Robert J.
Falk Anne-Marie
Qian Celine
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