Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1997-02-19
2001-08-21
Martin, Jill (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S003000, C800S021000, C800S022000, C800S025000, C435S455000, C435S463000, C435S462000, C435S320100, C435S325000
Reexamination Certificate
active
06278040
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to transgenic mice and uses therefor. In a particular aspect, the present invention relates to transgenic mice which are deficient in one or more members of the steroid/thyroid superfamily of receptors, as well as various uses therefor.
BACKGROUND OF THE INVENTION
The steroid/thyroid superfamily of receptors comprises a broad range of regulatory compounds which are involved in a wide range of regulatory processes. For example, retinoic acid (RA) and related vitamin A derivatives (retinoids) comprise a collection of molecules that serve as signals to trigger and modulate complex morphogenic events during vertebrate development. In addition, retinoids serve to maintain homeostasis in the adult. Retinoids display profound effects on cell differentiation and proliferation, and have been used extensively to influence differentiation in organ and cell culture systems (reviewed by Tabin in Cell 66:199-217 (1991); and Brockes in
Neuron
2:1285-1294 (1989)). Retinoids can block the effects of tumor promoters in cell culture and have been used in chemoprevention, as well as in the primary treatment, of certain solid tumors and leukemias in humans (see, for example, Hong and Itri, “Retinoids and Human Cancer” in
The Retinoids: Biology, Chemistry and Medicine,
2nd Edition, M. B. Sporn, A. B. Roberts and D. S. Goodman (eds.), Raven Press Ltd.: New York. (1994); and Warrell et al. in
New Engl. J. Med.
324:1385-1393 (1991)).
Exposure of vertebrate embryos to retinoic acid leads to a variety of teratogenic effects, depending on the time and dose of the exposure (see, for example, Linney and LaMantia, “Retinoid signaling in mouse embryos” in
Advances in Developmental Biology,
Vol. 3, in press (1994); and Morris-Kay (ed.) in
Retinoids in Normal Development and Teratogenesis,
Oxford University Press: Oxford (1992)). The most prominent target tissues include the heart, the axial skeleton, cranial and cardiac neural crest derived tissues, and the limbs. Paradoxically, vitamin A deficiency leads to an overlapping spectrum of defects, indicating a requirement for retinoids during normal development as well as a common target whose proper action is essential for the execution of developmental programs. A central question arising from these observations is how a simple molecule such as retinoic acid can lead to such diverse biological effects.
A great deal of this complexity can be explained by the observation that retinoid receptors are members of the nuclear receptor superfamily of ligand-dependent transcription factors (see, for example, Evans in
Science
240:889-895 (1988); and Green and Chambon in
Trends in Genet.
4:309-314 (1988)). Retinoid receptors comprise two distinct subfamilies composed of three retinoic acid receptors (RARs) and three evolutionarily distinct retinoid X receptors (RXRs) (see Mangelsdorf et al. in
Genes and Devel.
6:329-344 (1992) and references therein). The RARs and RXRs share overlapping ligand specificity, i.e., both receptors bind 9-cis retinoic acid with high affinity, whereas only the RARs bind all-trans retinoic acid (see Heyman et al. in Cell 68:397-406 (1992); and Levin et al. in
Nature
355:359-361 (1992)). It has been shown in vitro that RXRs are able to bind DNA as homodimers, whereas the RARs (as well as receptors for other hormones or hormone-like compounds, e.g., thyroid hormones (TRs), vitamin D (VDR), and peroxisome proliferators (PPARs)) form heterodimers with RXRs. Such heterodimers bind DNA in a highly cooperative fashion (see, for example, Yu et al. in
Genes and Dev.
6:1783-1798 (1991); Kliewer et al. in
Nature
355:446-449 (1992); Leid et al. in Cell 68:377-395 (1992); Marks et al. in
EMBO J.
11:1419-1435 (1992); Zhang et al. in Nature 355:441-446 (1992); and Kleiwer et al. in
Nature
358: 771-774 (1992)). The RXRs, therefore, apparently play a central role in mediating multiple hormonal signaling pathways.
Each RXR (and RAR) subtype is differentially expressed in a spectrum of tissues during normal embryonic development and beyond (see Mangelsdorf et al., supra). The RXR&agr; gene, for example, is abundant in the intestine, heart, muscle, liver, kidney, and skin of the adult, whereas the RXR&bgr; gene is expressed at a low level in nearly all tissues. RXR&ggr; shows the most restricted pattern of expression in both the embryo and adult, with highest levels in mesoderm and its derivatives and in parts of the nervous system.
To allow a more complete understanding of the diverse role of members of the steroid/thyroid superfamily of receptors (e.g., retinoid receptors) in development and/or physiology, it would be desirable to be able to correlate defects associated with ligand (e.g., retinoid) excess or deficiency with the presence or absence of individual receptor gene products.
BRIEF DESCRIPTION OF THE INVENTION
In accordance with the present invention, transgenic mice have been developed through the introduction of specific mutations into the germline thereof. Such mice enable the functional analysis of individual receptor genes to be carried out in vivo. For example, individual mutations of the RAR&agr; and RAR&ggr; genes have surprisingly been found not to be lethal in the embryonic stage. In fact, the resulting mutants display fairly subtle phenotypes.
In contrast, mutation of the RXR gene has been found to result in embryonic lethality, apparently due to hypoplastic development of the ventricular chambers of the embryonic heart. This mutation also results in strikingly delayed development of the embryonic liver. This delayed development is unlikely, however, to be causal to the embryonic lethality. These results provide the first genetic evidence for a role of RXRs in retinoid signaling and establish an essential role for this receptor in embryogenesis.
REFERENCES:
Evans, R.M., “The Steroid and Thyroid Hormone Receptor Superfamily”Science240:889-895 (1988).
Green and Chambon, “Nuclear receptors enhance our understanding of transcription regulation”Trends in Genetics4:309-314 (1992).
Kliewer et al., “Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors”Nature358:771-774 (1992).
Mangelsdorf et al., “Characterization of three RXR genes that mediate the action of 9-cis retinoic acid”Genes&Development6:329-344 (1992).
Miller-Hance et al., “In Vitro Chamber Specification during Embryonic Stem Cell Cardiogenesis”J. Biol. Chem.268:25244-25252 (1993).
O'Brien et al., “Positional specification of ventricular myosin light chain 2 expression in the primitive murine heart tube”Proc. Natl. Acad. Sci. USA90:5157-5161 (1993).
Schwab et al., “Human N-myc gene contributes to neoplastic transformation of mammalian cells in culture”Nature316:160-162 (1988).
Sucov et al., “RXR&agr; mutant mice establish a genetic basis for vitamin a signaling in heart morphogenesis”Genes&Development8:1007-1018 (1994).
Thomas and Capecchi, “Site-Directed Mutagenesis by Gene Targeting in Mouse Embryo-Derived Stem Cells”Cell51:503-512 (1987).
Yancopoulos et al., “N-myc can cooperate with ras to transform normal cells in culture”Proc. Natl. Acad. Sci. USA82:5455-5459 (1985).
Kubalak, et al., “Chamber Specification of Atrial Myosin Light Chain-2 Expression Precedes Septation during Murine Cardiogenesis”J. Biochem269:16961-16970 (1994).
Moreadith et al., J. Mol. Med., vol. 75, pp. 208-216, 1997.*
Lufkin et la., PNAS 90 :7225-7229 (1993).*
Li et al., PNAS 90: 1590-1594 (1993).*
Lohnes et al., Cell 73: 643-658 (1993).
Chien Kenneth R.
Evans Ronald M.
Sucov Henry M.
Gray Cary Ware & Freidenrich LLP
Martin Jill
Reiter Stephen E.
The Salk Institute for Biological Studies
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