Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1999-11-18
2003-06-10
Fredman, Jeffrey (Department: 1636)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S014000, C800S021000, C800S008000, C435S320100, C435S325000, C435S455000
Reexamination Certificate
active
06576813
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method to selectively disrupt a particular organ, tissue, or cell of an animal at a specific period, and an expression unit, vector, host cell, and kit that are utilized in the method. The invention also relates to a non-human animal in which a particular organ, tissue, or cell is disrupted by the method.
BACKGROUND ART
A normal function of a tissue or cell can be clarified by deleting the tissue or cell and examining the resulting effect. This procedure is performed by, for example, surgical ablation of a tissue or cell (including laser ablation) and specific expression of a toxin in a target cell (Palmiter, R. D. et al. (1987) Cell 50, 435-443; Breitman, M. L. et al. (1987) Science 238, 1563-1565; Borrelli, E. et al. (1988) Proc. Natl. Acad. Sci. USA 85, 7572-7576; Heyman, R. A. et al. (1989) Proc. Natl. Acad. Sci. USA 86, 2698-2702; Lowell, B. B. et al. (1993) Nature 3664 740-742; Ross, S. R. et al. (1993) Genes Dev. 7, 1318-1324).
When the target cells exist diffusely in the whole body, it is impossible to efficiently ablate only the target cells by the former method. When the target organ, tissue or cell is essential for normal development, the latter method results in death of the individual at embryo stage, and it is thus impossible to analyze animals after birth.
DISCLOSURE OF THE INVENTION
An objective of the present invention is to provide a method to specifically disrupt a particular organ, tissue, or cell at an arbitrary period.
Diphtheria toxin is a protein of 58 kDa and consists of two domains, fragment A and B. The fragment B domain binds to the precursor of heparin-binding EGF-like growth factor (HB-EGF)/diphtheria toxin receptor (DTR) which exists on the cell surface, and the toxin is incorporated into the cell via endocytosis. Upon fusion of the endosome with a lysosome, the conformation of the toxin is changed because of the effect of pH, and then the fragment A domain is translocated into the cytosol where the fragment inactivates peptide chain elongation factor (elongation factor 2) by ADP-ribosylating it and thus inhibits protein synthesis to bring the cell death (Honjo, T. et al. (1968) J. Biol. Chem. 243, 3553-3555; Pappenheimer, A. M. Jr. (1977) Annu. Rev. Biochem. 46, 69-94; Kohno, K. et al. (1986) Proc. Natl. Acad. Sci. USA 83, 4978-4982; Mekada, E. et al. (1988) J. Cell Biol. 107, 511-519; Moskaug, J. O. et al. (1991) J. Biol. Chem. 266, 2652-2659). The binding ability of diphtheria toxin to the precursor of HB-EGF differs among animal species. It is lower in mice than in humans or monkeys. Accordingly, a human and a monkey are sensitive to the toxin, whereas mice are not sensitive (Mitamura, T. et al. (1995) J. Biol. Chem. 270 (3), 1015-1019). Therefore, if a transgenic mouse is produced by introducing the human HB-EGF (hHB-EGF)/DTR gene inserted at the downstream of a promoter that functions specifically in a target organ, tissue, or cell so as to express the gene specifically there, the target can be disrupted at an arbitrary period by administrating diphtheria toxin to the mouse.
In fact, it has been shown that L cells, a mouse fibroblast insensitive to diphtheria toxin, become sensitive to the toxin when the hHB-EGF/DTR gene was introduced and expressed (Naglish, J. G. et al. (1992) Cell 69, 1051-1061).
The present inventors selected the enhancer/promoter of the albumin gene, which is specifically expressed in hepatic parenchymal cells (Gorski, K. et al. (1986) Cell 47, 767-776; Pinkert, C. A. et al. (1987) Genes Dev. 1, 268-276) as a specific promoter for a particular organ, tissue, or cell. The hHB-EGF/DTR gene was ligated to the downstream of the promoter, and transgenic mice were created by introducing the expression unit. Next, the inventors confirmed that the hHB-EGF/DTR gene was actually expressed specifically in the hepatic cells of the transgenic mice and examined the process of the disruption of hepatic parenchymal cells by administrating diphtheria toxin to the mice. As a result, the inventors found hepatic cells of the transgenic mice were specifically disrupted depending on the administration period of the toxin, and accomplished the present invention.
Thus, the present invention relates to a method for specifically disrupting a desired organ, tissue, or cell at a desired period by administrating a compound, and more specifically relates to:
(1) an expression unit comprising a gene encoding a receptor for a compound, which is essentially nontoxic to a host, at the downstream of a promoter functioning specifically in a particular organ, tissue, or cell, wherein said compound binds to said receptor artificially expressed in the host and shows a selective toxicity to the cell expressing said receptor;
(2) the expression unit according to (1), wherein said compound is diphtheria toxin, and said receptor for the toxin is the precursor of heparin-binding EGF-like growth factor (HB-EGF)/diphtheria toxin receptor;
(3) a vector comprising the expression unit of (1) or (2);
(4) a host cell carrying the expression unit of (1) or (2), or a vector of (3);
(5) a non-human animal carrying the expression unit of (1) or (2), or the vector of (3);
(6) a method for specifically disrupting a particular organ, tissue, or cell at a particular period in the animal of (5), wherein said method comprises administering a compound that is essentially nontoxic to the host at said particular period;
(7) the method of (6), wherein said animal is a mouse, and said compound is diphtheria toxin;
(8) a non-human animal, in which a particular organ, tissue, or cell is disrupted by the method of (6) or (7);
(9) a kit comprising the compound of (1) and a DNA comprising the expression unit of (1).
The invention relates to an expression unit comprising a DNA encoding a receptor for a compound, which is essentially nontoxic to the host, at the downstream of a promoter functioning specifically in a particular organ, tissue, or cell.
The promoter functioning specifically in a particular organ, tissue, or cell is not particularly limited. Examples of the promoter includes those of genes: the H-2 class II gene, specific for macrophages and dendritic cells; interferon &agr;, for granulocytes; interleukins 2, 4, and 5, for T lymphocytes; insulin, for &bgr; cells in pancreas; GDNF, for glia cells; immunoglobulin, for B lymphocytes; BDNF, for neurons; HGF, for hepatic parenchymal cells; THY-1, for T lymphocytes and neurons; T-cell receptor, for T lymphocytes; and hydroxymethylglutaryl coenzyme A reductase (HMG) and factor IX, for liver.
For example, the compound that is essentially nontoxic to a host includes diphtheria toxin when the host is a mouse. The compound is not particularly limited as long as it has no toxicity in hosts into which a receptor gene is not introduced, but has a selective toxicity to a particular organ, tissue, or cell of the hosts into which a receptor gene is introduced and expressed.
The receptor for a compound that is essentially nontoxic to the host is not particularly limited as long as it exhibits toxicity to a cell expressing the receptor by binding to the compound. However, a receptor whose expression itself has a bad influence on the cell is not preferable. When the compound is diphtheria toxin, a preferable receptor is the precursor of HB-EGF (heparin-binding EGF-like growth factor)/diphtheria toxin receptor.
A form of a DNA encoding the receptor is not limited as long as it encodes the receptor protein. The DNA includes cDNA, genomic DNA, and chemically synthesized DNA, etc. The DNA encoding the receptor can be prepared by a standard method (Molecular Cloning, 2nd edition, chapters 16 and 17, Cold Spring Harbor Laboratory Press).
The DNA can be inserted at the downstream of the promoter by a standard method (Molecular Cloning, 2nd edition, chapters 16 and 17, Cold Spring Harbor Laboratory Press). The resulting product can be an expression unit of the invention.
The invention also relates to a vector comprising the above expression unit.
The vector of the present invention is not particularly li
Clark & Elbing LLP
DNAVEC Research Inc.
Fredman Jeffrey
Kaushal Sumesh
LandOfFree
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