Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1998-01-26
2001-02-27
LeGuyader, John L. (Department: 1633)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S003000, C800S008000, C800S009000, C800S013000, C800S014000, C800S021000
Reexamination Certificate
active
06194633
ABSTRACT:
BACKGROUND OF THE INVENTION
Engagement of the T cell antigen receptor (TCR) results in the activation of protein tyrosine kinases (PTK) and the subsequent tyrosine phosphorylation of numerous proteins (Howe, L. R. and Weiss, A. (1995)
Trends Biochem. Sci.
20:59-64; see also Perlmutter, R. M. et al. (1993)
Annu. Rev. Immunol.
11:451-499; and Chan, A. C. et al. (1994)
Annu. Rev. Immunol.
12:555-592). Efforts to characterize substrates of the TCR induced PTK activity led to the cloning of a 76 kDa protein termed SLP-76 (for SH2-domain-containing Leukocyte Protein of 76 kDa). SLP-76 was originally identified based upon its ability to interact with the protein Grb2, an adaptor molecule involved in coupling signal transduction pathways (Motto, D. et al. (1994)
J. Biol. Chem.
269:21608-21613; Reif, K. et al. (1994)
J. Biol. Chem.
269:14081-14087; Buday, L. et al. (1994)
J. Biol. Chem.
269:9019-9023; and Sieh, M. et al. (1994)
Mol. Cell. Biol.
14:4435-4442).
Molecular cloning of SLP-76 cDNAs (human and mouse) revealed that the SLP-76 protein comprises an acidic amino-terminal region, a proline-rich central region and a carboxy-terminal SH2 domain (Jackman J. K. et al. (1995)
J. Biol. Chem.
270:7029-7032). Northern analysis demonstrated that SLP-76 mRNA is expressed exclusively in peripheral blood leukocytes, spleen and thymus (Jackman, J. K et al. (1995) supra). Insight into the function of SLP-76 in T cells came from experiments showing that overexpression of SLP-76 augments TCR-mediated signals that lead to the induction of IL-2 gene promoter activity (Motto, D. G. et al. (1996)
J. Exp. Med.
183:1937-1943; Wu, J. et al. (1996)
Immunity
4:593-602). Interestingly, three distinct regions of SLP-76 that are responsible for protein-protein interactions in T cells are required for the augmentation of IL-2 promoter activity by overexpression of SLP-76 (Fang, N. et al. (1996)
J. Immunol.
157:3769-3773; Wardenburg, J. B. et al. (1996)
J. Biol. Chem.
271:19641-19644; Musci, M. A. et al. (1997)
J. Immunol.
159:1639-1647). These data suggest that SLP-76 functions as a link between proteins that regulate signals generated by TCR ligation.
Certain SLP-76-associated proteins that are thought to participate with SLP-76 in transducing signals from the TCR to the nucleus have been identified. Examples include the protooncogene Vav, which associates with the amino-terminal acidic region of SLP-76 in a phosphotyrosine dependent manner (Wu, J. et al. (1996)
Immunity
4:593-602; Onodera, H. et al. (1996)
J. Biol. Chem.
271:22225-22230; Tuosto, L. et al. (1996)
J. Exp. Med.
184:1161-1167) and SLAP-130, a 130 kDa phosphoprotein that associates with the SH2 domain of SLP-76 and may act as a negative regulator of signal transduction (Musci, M. A. et al. (1997)
J. Biol. Chem.
272:11674-11677). However, the precise role of SLP-76 in cells of the hematopoietic lineage is unclear. Accordingly, model systems in which to assess the role of SLP-76 in hematopoietic developments, as well as the involvement of SLP-76 in disease states are needed.
SUMMARY OF THE INVENTION
This invention pertains to a nonhuman animal having somatic and germ cells in which at least one allele, and preferably both alleles, of an endogenous SLP-76 gene contains exogenous DNA that has been inserted into the endogenous SLP-76 gene such that expression of the endogenous SLP-76 gene is functionally disrupted. Accordingly, the invention provides viable animals having a mutated SLP-76 gene, and thus lacking SLP-76 activity. In a preferred embodiment, the exogenous DNA that is inserted into the SLP-76 allele comprises a selectable marker gene (such as a neomycin phosphotransferase gene).
Characterization of the phenotype of the SLP-76 deficient animals of the invention revealed that functional disruption of the SLP-76 gene results in a block in T cell development such that SLP-76 deficient animals are severely lacking in peripheral T cells. In contrast, B cell and macrophage development is not substantially affected, if at all, by functional disruption of the SLP-76 gene. Thus, the animals of the invention are characterized by:
(a) a substantially decreased percentage of mature T cells in peripheral blood as compared to a non-mutant animal of the same species;
(b) a substantially normal percentage of mature B cells in peripheral blood as compared to a non-mutant animal of the same species; and
(c) a substantially normal percentage of macrophages in peripheral blood as compared to a non-mutant animal of the same species.
In the nonhuman animal of the invention, the SLP-76 gene preferably is disrupted by homologous recombination between the endogenous allele and a mutant SLP-76 gene, or portion thereof, that has been introduced into an embryonic stem cell precursor of the animal. The embryonic stem cell precursor is then allowed to develop (i.e., by microinjection into a blastocyst and implantation of the blastocyst into a pseudopregnant foster animal), resulting in an animal having a functionally disrupted SLP-76 gene. The animal may have one SLP-76 gene allele functionally disrupted (i.e., the animal may be heterozygous for the mutation), or more preferably, the animal has both SLP-76 gene alleles functionally disrupted (i.e., the animal can be homozygous for the mutation). In one embodiment of the invention, functional disruption of both SLP-76 gene alleles produces animals in which expression of the SLP-76 gene product in cells of the animal is substantially reduced or substantially absent relative to non-mutant animals. In another embodiment, the SLP-76 gene alleles can be disrupted such that an altered (i.e., mutant) SLP-76 gene product is produced in cells of the animal. A preferred nonhuman animal of the invention having a functionally disrupted SLP-76 gene is a mouse.
The animals of the invention are useful, for example, as animal models of T cell immunodeficiency, as an in vivo system to evaluate the role of SLP-76 in various biological processes (e.g., T cell development, B cell development, macrophage development, platelet function, and the like), and as standard controls by which to evaluate the efficacy and side effects of putative SLP-76 inhibitors. The animals also can also be used to identify disease states for treatment with SLP-76 inhibitors. For example, the invention provides a method for identifying a disease condition treatable with a SLP-76 inhibitor which involves attempting to induce the disease condition in the animal of invention and determining the susceptibility or resistance of the animal to the disease condition. Resistance of the SLP-76 deficient animal to the disease condition, relative to a nonmutant animal of the same species, is indicative that the disease condition is treatable with a SLP-76 inhibitor. One can attempt to induce the disease condition in the animal by, for example, administering to the animal a stimulus that induces the disease condition in a nonmutant animal of the same species or by breeding the SLP-76 deficient animal with a second animal that is susceptible to the disease condition.
Another aspect of the invention pertains to a nonhuman animal having a functionally disrupted endogenous SLP-76 gene but which also carries in its genome, and expresses, a SLP-76 transgene randomly integrated into the genome of the animal. In one embodiment, the transgene encodes a SLP-76 protein from the same species as the animal (e.g., a SLP-76 deficient mouse can be reconstituted with a mouse SLP-76 transgene), although expression of the transgene can be directed to a particular cell population. In another embodiment, the SLP-76 transgene encodes a heterologous SLP-76 protein (i.e., a SLP-76 protein from another species). Preferably, the animal is a mouse and the heterologous SLP-76 is a human SLP-76. An animal of the invention which has been reconstituted with human SLP-76 can be used to identify agents that inhibit human SLP-76 in vivo. For example, an agent to be tested can be administered to such an animal and the activity of human SLP-76 in the animal can be measured. Dec
Clements James L.
Koretzky Gary A.
Williamson Roger
Hanley, Esq. Elizabeth A.
Lahive & Cockfield LLP
Lauro, Esq. Peter C.
LeGuyader John L.
University of Iowa Research Foundation
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