Vitamin D receptor ablated mice

Details Vitamin D receptor ablated mice Vitamin D receptor ablated mice

1998-08-28

2002-02-26

Clark, Deborah J. R. (Department: 1633)

Multicellular living organisms and unmodified parts thereof and

Method of using a transgenic nonhuman animal in an in vivo...

C800S008000, C800S009000, C800S013000, C800S014000, C800S018000, C435S325000, C435S354000, C435S455000, C435S463000, C514S880000

Reexamination Certificate

active

06350932

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to animals in which the vitamin D receptor is misexpressed and methods of using such animals or cells derived therefrom, e.g., in methods of evaluating treatments for skin disorders, immune disorders and proliferation-related disorders.
BACKGROUND OF THE INVENTION
The vitamin D receptor (VDR), is a nuclear receptor which heterodimerizes with the retinoid X receptor and interacts with specific DNA sequences on target genes, regulating their expression. The VDR is evolutionary well conserved and is expressed early in development in amphibians (Li Y. et al., (1997) Endocrinology 138:2347-2353), mammals (Johnson J. A. et al., (1996) J. Bone Miner. Res. 11:56-61), and birds (Elaroussi M. A. et al., (1994) Biochim. Biophys. Acta 1192:1-6; and Tuan R. S. and Suyama E., (1996) J. Nutr. 126:1308S-1316S). The VDR is expressed in tissues involved in mineral ion homeostasis, such as the intestine, the skeleton, and the parathyroid glands, as well as in tissues not thought to play a role in mineral ion homeostasis, such as the epidermis and the pancreas (Stumpf W. E. et al., (1979) Science 206:1188-1190).
Its principal ligand, 1,25-dihydroxyvitamin D is a steroid hormone that plays a role in mineral ion homeostasis. Insights into the physiological actions of 1,25-dihydroxyvitamin D have been obtained from studies in vitamin D deficient animals (Dostal L. A. and Toverud S. U., (1984) Am J. Physiol. 246:G528-G534; Halloran B. P. and DeLuca H. F., (1981) Arch. Biochem. Biophys. 209:7-14; Mathews C. H. E. et al., (1986) Am. J. Physiol. 250:E725-E730; Miller S. C. et al., (1983) Calcif. Tissue Int. 35:455-460; and Underwood J. L. and DeLuca H. F., (1984) Am. J. Physiol. 246:E493-E498) as well as in humans with VDR mutations (Balsan S. et al., (1986) J. Clin. Invest. 77:1661-1667; and Beer S. et al., (1981) Clin. Endocrinol. 14:395-402). These investigations have demonstrated that 1,25-dihydroxyvitamin D plays an important role in intestinal calcium absorption and that animals lacking in active hormone or its nuclear receptor develop hypocalcemia, rickets, osteomalacia, and hyperparathyroidism.
1,25-dihydroxyvitamin D is also believed to inhibit parathyroid hormone synthesis in the parathyroid gland (Silver J. et al., (1985) Proc. Natl. Acad. Sci. USA 82:4270-4273), blocking interleukin-2 production in activated lymphocytes (Manolagas S. C. et al., (1990) Kidney Int. 29:S9-S16), stimulating insulin secretion in the pancreas (Norman A. W. et al., (1980) Science 209:823-825), and decreasing proliferation and inducing differentiation of keratinocytes in the epidermis (Bikle D. D. et al., (1988) Endocrinology 124:655-660).
SUMMARY OF THE INVENTION
In general, the invention features, a non-human animal, in which the gene encoding the vitamin D receptor is misexpressed.
In preferred embodiments the animal, which is preferably a transgenic animal, is a mammal, e.g., a nonhuman primate or a swine, e.g., a miniature swine, a monkey, a goat, or a rodent, e.g., a rat, but preferably a mouse.
In preferred embodiments, expression of the gene encoding the vitamin D receptor is decreased as compared to the wild-type mouse. For example, the levels of the vitamin D receptor can be suppressed by, at least, 50, 60, 70, 80 or 90% or 100% as compared to the wild-type mouse.
In preferred embodiments, misexpression of the gene encoding the vitamin D receptor is caused by disruption of the vitamin D receptor gene. For example, the vitamin D receptor gene can be disrupted through removal of DNA encoding all or part of the receptor, e.g., removal of all or part of a zinc-finger domain, e.g., the second zinc-finger of the DNA-binding domain of the receptor.
In preferred embodiments, the animal can be heterozygous or homozygous for a misexpressed VDR gene, e.g., it can be a transgenic animal heterozygous or homozygous for a VDR transgene.
In preferred embodiments, the animal is a transgenic mouse with a transgenic disruption of the VDR, preferably an insertion or deletion, which inactivates the gene product.
In another aspect, the invention features, a nucleic acid sequence which, when introduced into an animal or cell, results in the misexpression of the VDR gene in the animal or cell. In preferred embodiments, the nucleic acid sequence, includes a VDR sequence which includes a disruption, e.g., an insertion or deletion and preferably the insertion of a marker sequence. For example, nucleic acid sequence can be the targeting construct, shown in FIG.
1
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In another aspect, the invention features, a method of evaluating a treatment for a skin disorder. The method includes: administering the treatment to a VDR misexpressing animal or a cell therefrom; and determining the effect of the treatment on a parameter related to the skin disorder, to thereby evaluate the treatment for the skin disorder. The method may be performed in vivo or in vitro.
In preferred embodiments, the animal or cell is an animal or cell described herein.
In preferred embodiments, the method further includes determining the effect of the treatment on the levels, e.g., plasma levels, of calcium in the animal or cell.
In preferred embodiments, the skin disorder is a proliferative skin disorder. For example, the skin disorder can be a hyperproliferative skin disorder, e.g., psoriasis, squamus cell carcinoma, alopecia and the like.
In preferred embodiments, the effect of the treatment on the proliferative skin disorder can be determined by measuring such parameters as the thickness of the keratinocyte layer or the number and size of papules present on the skin of the animal; cell growth; tumor growth, weight or invasiveness; life span; tissue morphology; weight; or the expression of a gene related to cell proliferation.
In preferred embodiments, the effect of the treatment on alopecia can be determined by measuring such parameters as hair growth (in terms of the number, thickness, or growth rate of hairs), hair follicle morphology, or the pattern of hair growth.
In preferred embodiments, the method uses a transgenic mouse in which the expression of the VDR is inhibited.
In preferred embodiments, the method uses a cell derived from a transgenic mouse in which the expression of the VDR is inhibited.
In another aspect, the invention features, a method of evaluating a treatment for an immune disorder or condition. The method includes: administering the treatment to a VDR misexpressing animal or a cell therefrom; and determining the effect of the treatment on a parameter related to the immune disorder or condition, to thereby evaluate the treatment for the immune disorder or condition. The method may be performed in vivo or in vitro.
In preferred embodiments, the animal or cell is an animal or cell described herein.
In preferred embodiments, the method further includes determining the effect of the treatment on the levels, e.g., plasma levels, of calcium in the animal or cell.
In preferred embodiments, the effect of the treatment on the immune disorder or condition can be determined by measuring a parameter such as: the presence, function, or morphology of T cells or their progenitors; the presence, function, or morphology of B cells or their progenitors; the presence, function, or morphology of natural killer cells or their progenitors; resistance to infection; life span; body weight; the presence, function, or morphology of tissues or organs of the immune system; or the expression of a gene related to an immune disorder or condition.
In preferred embodiments, the method uses a transgenic mouse in which the expression of the VDR is inhibited.
In preferred embodiments, the method uses a cell derived from a transgenic mouse in which the expression of the VDR is inhibited.
In another aspect, the invention features, a method of evaluating a treatment for promoting acceptance of a graft. The method includes: providing an animal, in which the gene encoding the vitamin D receptor is misexpressed; (optionally) introducing a graft tissue into the animal; administering the treatment to the animal; and determining the effect of

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