Multicellular living organisms and unmodified parts thereof and – Method of using a transgenic nonhuman animal in an in vivo...
Reexamination Certificate
2001-07-11
2003-11-25
Paras, Peter (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of using a transgenic nonhuman animal in an in vivo...
C800S018000, C800S021000, C800S025000, C435S325000, C435S354000, C536S023100
Reexamination Certificate
active
06653526
ABSTRACT:
BACKGROUND OF THE INVENTION
The ubiquitin-specific protease 16s are a family of enzymes that cleave ubiquitin from ubiquitinated protein substrates, and are important in many cellular processes. Ubiquitin is a highly conserved polypeptide found in all eukaryotes, and its major function is to target proteins for complete or partial degradation by a multisubunit protein complex called the proteasome. The ubiquitin-dependent proteolyic pathway is mediated by a diverse array of enzymes and is one of the major routes by which intracellular proteins are selectively destroyed. (See, e.g., Hochstrasser M.,
Current Opinion In Cell Biology
4:1024-1031 (1992)).
In eukaryotes, conjugation to ubiquitin polymers often targets a protein for destruction. As a part of this process, dubiquitinating enzymes disassemble ubiquitin polymers or ubiquiting-substrate conjugates. For example, the dubiquitinating enzyme, UbpA, is required for development of Dictoyostelium. More particularly, specific developmental transitions in Dictyostelium require degradation of specific proteins that require the disassembly of polyubiquitin chains by UbpA. (See, e.g., Lindsey et al.,
Journal of Biological Chemistry
273:29178 (1998)).
Deubiquitinating enzymes serve a number of functions in the ubiquitin-dependent proteolytic pathway. (See, e.g., Hochstrasser (1992), supra; Rose, I. A., In
Current Communications In Molecular Biology: The Ubiquitin System
, Schlesinger and Hershko (eds.) Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1988)). First, the enzymes cleave ubiquitin from biosynthetic precursors occurring either as a series of ubiquitin monomers in head-to-tail linkage or as fusions to certain ribosomal proteins (See, e.g., Finley & Chau,
Annual Review of Cell Biology
7:25-69 (1991)). Secondly, ubiquitin is recycled from intracellular conjugates, both to maintain adequate pools of free ubiquitin, and to reverse the modification of inappropriately targeted proteins. Lastly, deubiquitinating reactions are important to the degradation of ubiquitinated proteins by the 26S proteasome, a complex ATP-dependent enzyme.
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 ubiquitin-specific protease 16 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 a ubiquitin-specific protease 16 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 ubiquitin-specific protease 16 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 ubiquitin-specific protease 16 gene. The transgenic animals of the present invention include transgenic animals that are heterozygous and homozygous for a mutation in the ubiquitin-specific protease 16 gene. In one aspect, the transgenic animals of the present invention are defective in the function of the ubiquitin-specific protease 16 gene. In another aspect, the transgenic animals of the present invention comprise a phenotype associated with having a mutation in a ubiquitin-specific protease 16 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 ubiquitin-specific protease 16 gene.
The present invention further provides a method of identifying agents having an effect on a ubiquitin-specific protease 16 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 a ubiquitin-specific protease 16 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 ubiquitin-specific protease 16 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 ubiquitin-specific protease 16 gene sequence is under the control of an inducible promoter. Also provided are methods of identifying agents that interact with the ubiquitin-specific protease 16 gene, comprising the steps of contacting the ubiquitin-specific protease 16 gene with an agent and detecting an agent/a ubiquitin-specific protease 16 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 ubiquitin-specific protease 16 gene, and more particularly, to a disruption in the expression or function of the ubiquitin-specific protease 16 gene. In a preferred embodiment, methods of the present invention involve treating diseases or conditions associated with a ubiquitin-specific protease 16 gene's expression or function, including administering to a subject in need, a therapeutic agent which effects a ubiquitin-specific protease 16 expression or function.
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 ubiquitin-specific protease 16 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 polym
Deltagen Inc.
Driscoll Robert J.
Lapiz Mariette A.
Paras Peter
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