Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
2001-07-06
2004-04-06
Falk, Anne-Marie (Department: 1636)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S021000, C435S325000
Reexamination Certificate
active
06717032
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) includes 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.
Recently, a novel orphan GPCR termed the DEZ receptor (aka chemokine-like receptor 1, or CMKLR1) 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 cds for the murine DEZ orphan receptor, or CMKLR1, has been deposited in GenBank (Accession number: U79525; GI/NID number: 1732346).
Given the importance of GPCRs, particularly the DEZ orphan receptor, 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 DEZ receptor 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 DEZ receptor 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 DEZ receptor 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 DEZ receptor gene. The transgenic animals of the present invention include transgenic animals that are heterozygous and homozygous for a mutation in the DEZ receptor gene. In one aspect, the transgenic animals of the present invention are defective in the function of the DEZ receptor gene. In another aspect, the transgenic animals of the present invention comprise a phenotype associated with having a mutation in a DEZ receptor gene.
The present invention also provides methods of identifying agents capable of affecting a phenotype of 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 DEZ receptor gene.
The present invention further provides a method of identifying agents having an effect on DEZ receptor 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 DEZ receptor 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 DEZ receptor 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 DEZ receptor gene sequence is under the control of an inducible promoter. Also provided are methods of identifying agents that interact with the DEZ receptor gene, comprising the steps of contacting the DEZ receptor gene with an agent and detecting an agent/DEZ receptor gene 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 DEZ receptor gene, and more particularly, to a disruption in the expression or function of the DEZ receptor gene. In a preferred embodiment, methods of the present invention involve treating diseases or conditions associated with a disruption in the DEZ receptor gene's expression or function, including administering to a subject in need, a therapeutic agent that effects DEZ receptor 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 DEZ receptor gene, DEZ receptor 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 DEZ receptor 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 seque
Deltagen, Inc
Driscoll Robert J.
Falk Anne-Marie
Qian Celine
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