Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1998-04-16
2001-07-03
Hauda, Karen M. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S013000, C800S021000, C800S025000, C435S320100, C536S023100
Reexamination Certificate
active
06255555
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an animal model for human diseases. More particularly, it relates to a transgenic mouse usable as an animal model for human acquired immunodeficiency syndrome (AIDS).
2. Disclosure of the Related Art
Human immunodeficiency virus (HIV) infection has spread throughout the world since 1981 when a patient with this infection was reported at the first time in the United State, and up to date, persons infected with HIV are presumed to exceed twenty millions. HIV infects human CD4
+
blood cells (i.e., helper T cell and macrophage) predominantly. The HIV infectious disease causes AIDS, which is characterized by decrease in CD4
+
T cells, after the latent period, thereby leading to death; that is, it is a disease that the prognosis is bad. Currently, no effective methods for prevention and treatment (e.g., vaccines) have been established, and the mechanism that HIV causes immunodeficiency has not yet been clarified.
One of the causes that make the solution of those problems late is that there exists no suitable small animal model for HIV infection. Anthropoids such as chimpanzees and monkeys have been utilized as animal models for HIV infection, whereas there are great demands on small animal models for HIV infection due to expensive management cost and limited facility.
Recently, the mouse that human blood cells have been transplanted in a hereditary immunodeficiency mouse (SCID mouse) so that the transplanted human blood cells could be infected with HIV has been developed as an animal model for HIV infection and has been used by some researchers (J. M. McCune et al., Science, 247:564-566, 1990).
However, when this mouse is used as an animal model for HIV infection, human blood cells must be transplanted in every individuals and HIV must be allowed to infect following settlement of the transplanted blood cells, i.e., several weeks after transplantation. Although SCID mouse is used to prevent the graft versus host (GVH) response following transplantation, the settlement of the transplanted human blood cells is insufficient or it does not occur sometimes. Thus, in cases where the SCID mouse is utilized in preparation of animal models for HIV infection, there are problems that preparation of such animal models needs the skill; that traits of individual mice are not constant with reflecting the efficiency of transplantation; and that no immunodeficiency is caused by HIV because HIV does not infect murine lymphocytes.
For the above reasons, there are great demands on small animal models for HIV infection, which are infected with HIV hereditarily; have constant traits; and are able to develop immunodeficiency seen in HIV infected patients.
In this situation, it has been reported in 1996 that when T-cell line tropic HIV strain invades T cells, fusin (or CXCR4) which is a cell surface protein of T lymphocytes is required as well as CD4 which is a other cell surface membrane protein of T lymphocytes (Y. C. Feng et al., Science (Wash. D.C.), 272:872-877, 1996; and J. F. Berson et al., J. Virol., 70:6288-6295, 1996).
The present inventor has continued to study actively in order to solve the above mentioned problems and, as a result, succeeded in production of a transgenic mouse in which at least two human T cell surface proteins are coexpressed on the surface of T lymphocytes of the mouse.
SUMMARY OF THE INVENTION
The present invention provides a transgenic mouse capable of pressing at least two cell surface membrane proteins of human T lymphocytes.
According to an embodiment of the present invention, the cell surface membrane proteins of human T lymphocytes are associated with HIV infection and are preferably CD4 and fusin (CXCR4). Genes encoding the cell surface membrane proteins of human T lymphocytes are integrated into the same chromosome of the mouse, preferably chromosomes in both gonocyte and somatic cells, and they are characterized by coexpression on the surface of murine CD4
+
T lymphocytes. In addition, the trait of expressing the cell surface membrane proteins of human T lymphocytes can be inherited from the transgenic mouse to its progeny.
According to a preferred embodiment of the present invention, there is provided a transgenic mouse in which human CD4 and fusin are coexpressed in CD4
+
T lymphocytes.
The present invention also provides a transgene comprising human fusin gene or human CD4 gene.
One of the transgenes comprises a transcriptional control region of murine CD4 gene, human fusin cDNA, and poly A addition signal in order in 5′→3′. The transcriptional control region may comprise, for example, murine CD4 enhancer, promoter, exon I, intron I, and part of exon II. Particularly, the transgene is Construct a shown in
FIG. 1
a.
Theother transgene comprises in 5′→3′: a murine CD4 enhancer, and a modified human genomic CD4 gene with a modified human CD4 intron III in order. The modified human genomic CD4 gene preferably comprises about 3 kb region upstream of the human genomic CD4 gene and about 3 kb region downstream of the same. The modified human CD4 intron III is preferably composed of about 1.5 kb region at the 5′-side of human CD4 intron III and about 3 kb region at the 3′-side of the same. In other embodiment, the transgene is shown in
FIG. 1
b.
The present invention further provides a method for preparing a transgenic mouse, which comprises mixing together at least two different transgenes that comprise genes encoding at least two cell surface membrane proteins of human T lymphocytes; injecting the mixture into a murine fertilized egg; transplanting the obtained fertilized egg in a foster parent female mouse; allowing the transplanted mouse to breed; and selecting offspring mice that are able to express the at least two cell surface membrane proteins of human T lymphocytes. In this method, the cell surface membrane proteins of human T lymphocytes and the transgenes are both as defined above, and the former are particularly associated with HIV infection: human CD4 and human fusin in accordance with the embodiment of the present invention.
REFERENCES:
patent: 2 692 435 (1993-12-01), None
patent: WO 94 06908 (1994-03-01), None
patent: WO 94 28915 (1994-12-01), None
patent: WO 97 08303 (1997-03-01), None
patent: WO 97 28258 (1997-08-01), None
Berson et al., “A Seven-Transmembrane Domain Receptor Involved in Fusion and Entry of T-Cell-Tropic Human Immunodeficiency Virus Type 1 Strains”, J. Virol., 70(9):6288-6295 (1996).
Feng et al., “HIV-1 Entry Cofactor: Functional cDNA Cloning of a Seven-Transmembrane, G Protein -Coupled Receptor”, Science, 272:872-877 (1996).
Hodge et al., “Humans with OKT4-Eptitope Deficiency Have a Single Nucleotide Base Change in the CD4 Gene, Resulting in Substitution of TRP240for ARG240”, Human Immunol., 30:99-104 (1991).
Kileen et al., “Regulated Expression of Human CD4 Rescues Helper T Cell Development in Mice Lacking Expression of Endogenous CD4”, Embo J., 12(4):1547-1553 (1993).
MacKay, “Chemokine Receptors and T Cell Chemotaxis”, J. Exp. Med., 184:799-802 (1996).
McCune et al., “Suppression of HIV Infection in AZT-Treated SCID-hu Mice”, Science, 247:564-566 (1990).
Murphy, Annu. Rev. Immunol., “The Molecular Biology of Leukocyte Chemoattractant Receptors”, 12:593-633 (1994).
Nomura et al., “Molecular Cloning of cDNAs Encoding a LD78 Receptor and Putative Leukocyte Chemotactic Peptide Receptors”, Int'l. Immunol., 5(10):1239-1249 (1993).
Oberlin et al., “The CXC Chemokine SDF-1 is the ligand for LESTR/fusin and Prevents Infection by T-Cell-Line-Adapted HIV-1”, Nature, 382:833-835 (1996).
Sawada et al., “A Lineage-Specific Transcriptional Silencer Regulated CD4 Gene Expression During T Lymphocyte Development”, Cell, 77:917-929 (1994).
Sawada et al., “Identification and Characterization of a T-Cell-Specific Enhancer Adjacent to the Murine CD4 Gene”, Mol. Cell. Biol., 11(11):5506-5515 (1991).
Unutmaz et al., “Expression Pattern of HIV-1 Coreceptors on T Cells: Implications for Viral
Beckerleg Anne Marie S.
Finnegan Henderson Farabow Garrett & Dunner
Hauda Karen M.
Japan Science and Technology Corporation
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