Multicellular living organisms and unmodified parts thereof and – Method of using a transgenic nonhuman animal in an in vivo...
Reexamination Certificate
1999-08-02
2003-07-01
Priebe, Scott D. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of using a transgenic nonhuman animal in an in vivo...
C800S003000, C435S455000
Reexamination Certificate
active
06586655
ABSTRACT:
FIELD OF THE INVENTION
This invention pertains to the field of transgenic non-human animals. In particular this invention pertains to transgenic animals that express human steroid hormone receptor genes in desired tissues, such as the epithelium, and to methods of screening potential therapeutics for activity at steroid hormone receptors.
BACKGROUND OF THE INVENTION
Steroid hormones are secreted by the adrenal cortex, testis, ovary and placenta and include the androgens (such as testosterone), estrogens (such as estradiol and estrone), glucocorticoids (cortisone, corticosterone, and cortisol), mineralocorticoids (primarily aldosterone), and progestogens (primarily progesterone). Steroid hormones regulate proliferation and differentiation in target cells within the reproductive tract, mammary gland, and peripheral tissues such as the bones, heart, blood vessels, and hair follicles (for a review, see Yamashita et al.,
Localization and functions of steroid hormone receptors,
1998,
Histol. Histopathol.
13(1):255-70).
Steroid hormones are highly lipophilic and act through nuclear receptors rather than through receptors on the plasma membrane. Steroid hormone receptors have been shown to support the development of cancer in the breast, prostate, uterus, cervix, and ovaries. In addition, steroid hormone receptors appear to prevent osteoporosis of the bones, to prevent atherosclerosis of the coronary arteries of the heart, and to mediate baldness in men. Therefore, steroid hormone nuclear receptors and other gene products that are involved in steroid hormone metabolism are attractive targets for the development of therapeutics that address the treatment of reproductive cancers and conditions such as osteoporosis, atherosclerosis, and baldness.
Steroid hormone receptors are part of a family of nuclear receptors which contain a hormone-biding region and a DNA-binding region, and thereby act as transcription enhancers. Upon binding to their specific ligands, nuclear receptors interact directly with regions of DNA in order to influence transcription of genes regulating hormonal activity. See, Ribeiro et al.,
The nuclear hormone receptor gene superfamily,
1995,
Annual Rev Med.
46:443-53. There is a growing list of drugs that either bind directly to steroid hormone receptors or modulate steroid hormone metabolism. Drugs that bind directly to the nuclear receptors include tamoxifen, an anti-estrogen used in treatment of breast cancer, and raloxifene, used in prevention of osteoporosis. Drugs that modulate sex steroid metabolism include aromatase inhibitors, used to treat breast cancer and possibly prostate cancer, and finasteride (Propecia™), used in the treatment of hair loss.
Models of targeted expression of oncogenes and growth factors to the epidermis of transgenic mice have been described. These models have used keratin promoters to target the expression of foreign DNA. The basal cell specific keratin-14 (K14) promoter has been used to express growth factors and oncogenes in transgenic animals as models for the development of specific carcinomas (Vassar et al., 1991,
Cell
64:365-380; U.S. Pat. No. 5,698,764). There is currently no transgenic model for the expression of human nuclear receptors in the epithelium.
Despite the existence of several drugs that either bind directly to steroid hormone receptors or modulate steroid hormone metabolism, there is a further need to develop models for assaying and testing the site of action and the efficacy of drugs that act to modulate nuclear receptor activity and metabolism. The present invention addresses these and other needs.
SUMMARY OF THE INVENTION
The present invention provides transgenic non-human animals exhibiting a detectable phenotype, typically epidermal hyperplasia, caused by a steroid hormone receptor gene operably linked to a promoter which directs expression of the steroid receptor gene in the epithelium in the non-human animal, preferably a mouse. The steroid receptor gene is preferably a human estrogen receptor gene. The promoter used to drive expression of the steroid receptor gene is preferably a keratin-14 promoter directing expression of the steroid receptor in a basal keratinocyte.
The animal may further comprise a &bgr;-galactosidase gene operably linked to a promoter which directs expression of the &bgr;-galactosidase gene in an epithelial cell. If the steroid hormone receptor gene is expressed in a basal keratinocyte, the &bgr;-galactosidase gene is also preferably expressed in a basal keratinocyte.
The invention also provides DNA constructs comprising an expression cassette including a steroid hormone receptor gene operably linked to a promoter which directs expression of the steroid hormone receptor in the epithelium, preferably in a basal keratinocyte. The promoter is preferably the keratin-14 promoter.
The invention further provides methods of testing a composition for the ability to modulate steroid hormone receptor activity. The methods comprise providing a transgenic non-human animal comprising a human steroid hormone receptor gene operably linked to a keratin-14 promoter which directs expression of the gene in an epithelial cell, administering the composition to the non-human animal, and detecting changes in the epithelial cell of the non-human animal.
Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.
As used herein, “non-human animals” include, for example, mammals such as non-human primates, ovine, canine, bovine, rattus and murine species, as well as rabbits and the like. Preferred non-human animals are selected from the rodent family, including rat, guinea pig and mouse, most preferably mouse.
As used herein, a “steroid” or “steroid hormone” refers to any one of a group of biologically active compounds synthesized from cholesterol that contain a cyclopentanoperhydrophenanthrene nucleus. A “steroid receptor” or “steroid hormone receptor” is a nuclear receptor that binds a steroid or steroid hormone described herein.
As used herein, the term “steroid hormone receptor gene” or “steroid receptor gene” refers to a nucleotide sequence, or any subsequence thereof, that encodes a steroid receptor described herein or that encodes a gene product exhibiting DNA-binding and steroid hormone-binding activity, in vitro or in vivo; and any conservatively modified variants thereof. Also explicitly included within this definition are both wild-type and mutant genes (e.g. mutant steroid hormone receptor genes isolated from cancer cells) that may or may not have altered activity as compared to wild-type genes.
The term “estrogen receptor” refers to a known nuclear receptor having a predicted molecular weight of about 66-kd, that is activated by estrogenic steroid hormones such as estradiol. The active form of the protein enhances expression of genes involved in the formation of secondary sexual characteristics in mammalian females. An estrogen receptor can be an allele, polymorphic variant, interspecies homolog, or any subsequence thereof that exhibits estrogenic steroid hormone-binding activity.
As used herein, “estrogen receptor gene” is a wild-type or mutant nucleotide sequence that encodes an estrogen receptor described herein, and conservatively modified variants thereof. An example of an estrogen receptor gene is described in Greene et al., 1986,
Science
231(4742):1150-4. One of ordinary skill in the art will recognize that certain modifications, additions, and deletions may be made to the estrogen receptor gene sequence which will not affect the function or activity of the gene product. Such variants are included within this definition. An example of a mutant estrogen receptor gene is K206A,
Anderegg Birgit
Arbeit Jeffrey M.
Kushner Peter J.
Uht Rosalie M.
Priebe Scott D.
The Regents of the University of California
Townsend and Townsend and Crew
Wilson Michael C.
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