Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1998-03-17
2001-01-30
LeGuyader, John L. (Department: 1633)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S003000, C800S008000, C800S009000, C800S010000, C800S013000, C800S014000, C435S325000
Reexamination Certificate
active
06180849
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to transgenic animals.
BACKGROUND OF THE INVENTION
Several families of RNA-binding proteins are involved in a variety of fundamental cellular processes including RNA splicing, polyadenylation, RNA transport, and translation. Members of the RNA recognition motif (RRM)/ribonucleoprotein (RNP) family contain one to four approximately 100 amino acid long RRM domains in association with various auxiliary domains. TIAR is a RRM protein that is comprised of three N-terminal RRM domains and a C-terminal auxiliary domain (Beck et al., Nucleic Acid Research 24: 3829-3835, 1996; Dember et al., J. Biol. Chem 271: 2783-2788, 1996; Taupin et al., Proc. Natl. Acad. Sci. USA 92: 1629-1633, 1995; Tian et al., Cell 67: 629-639, 1991). Murine TIAR is predominantly expressed in brain, testis, and spleen. At least two isoforms of the protein are generated by alternative splicing (FIG.
1
).
SUMMARY OF THE INVENTION
The invention features transgenic non-human mammals (e.g., rodents such as mice and rats) all diploid cells of which contain a mutation in one or both alleles of the endogenous TIAR gene. The mutation can, for example, be a deletion, an insertion, or a nucleotide substitution. It can also be a translocation involving one or more exons, introns or transcription regulatory regions (e.g., a promoter) of the gene. If the mutation reduces the expression or activity level of the protein encoded by the mutated gene (all isoforms included) by more than 80% relative to the unmutated TIAR gene, the mutation is called a null mutation, and the mouse harboring the mutation is a knockout mouse. A homozygous TIAR null mutation has been found to cause partial lethality, infertility, obesity and neurological disorders in mice.
Also embraced by the present invention are cell lines derived from these transgenic non-human mammals. These cell lines can be of any cell origin (e.g., primordial germ cell, fibroblast, ovarian cell, or neuron).
Transgenic mice homozygous for TIAR null mutation are infertile. One cause of infertility in these mice is the degeneration of primordial germ cells (PGCs). Thus, within the present invention is also a method of identifying fertility-enhancing compounds by use of TIAR-deficient transgenic mice. In this method, an appropriate amount of a test compound is first administered to TIAR-deficient mice. Survival of PGCs in these mice is then examined. Enhanced survival of these PGCs as compared to PGCs of control TIAR-deficient mice which have not been exposed to the test compound is an indication that the test compound is capable of enhancing fertility of a mammal.
Fertility-enhancing compounds can also be identified by use of PGCs derived from TIAR-deficient mice (e.g., in the form of primary cell cultures or cell lines). In particular, the PGCs can be obtained from these mice (e.g., from the genital ridges of these mice) at around day 11. To test a compound for its fertility-enhancing ability, the cultured PGCs are first contacted with an appropriate amount of the compound for an appropriate duration of time. Subsequently, the growth rate, life span, differentiation, or any other appropriate growth or metabolic parameter of the PGCs is examined by standard techniques. For instance, growth rate can be determined by the amount of radioactive nucleotides incorporated into replicating genomic DNA, or by the number of cells generated by cell division. Differentiation of these PGCs, on the other hand, can be assessed by change of protein expression pattern or morphology known to occur during normal PGC development. Increased growth rate, life span, and/or differentiation of the PGCs as compared to control PGCs which have not been treated with the test compound is an indication that the compound is capable of enhancing fertility of a mammal.
Female TIAR-deficient mice develop ovarian sex cord stromal tumors as they age. Thus, ovarian sex cord stromal tumor cells derived from these mice can be used to screen for chemotherapeutic compounds useful in treating ovarian cancer such as ovarian sex cord stromal tumors. To do so, these cells are contacted with a candidate compound, and the growth rate of the cells is examined, where a decrease in the growth rate as compared to an untreated control indicates that the compound inhibits the growth of ovarian sex cord stromal tumor cells. The compound can also be administered directly to a female TIAR-deficient mouse, and a delay, prevention, or even reversal of the development of ovarian sex cord stromal tumor indicates that the compound can be used to treat or prevent ovarian sex cord stromal tumors.
TIAR-deficient mice are underweight at birth but become obese at the age of approximately 2 to 4 months. The leptin level in these mice is abnormally high, and the mice tend to consume a larger amount of food than normal mice. Thus, to identify compounds that are capable of reducing body weight of an obese mammal, one can administer a test compound to TIAR-deficient transgenic mice after they become obese. After an appropriate duration of time (e.g., one to four weeks), the average body weight of these mice is compared to that of control mice which have not been exposed to the compound. A significant decrease (e.g., at least 20%, or even 50%) of the average body weight as compared to control is an indication that the compound is useful in reducing the body weight of an obese mammal. Similarly, the ability of a test compound to prevent excessive weight gain in these mice can be tested by beginning administration prior to the onset of obesity. Alternatively, one can determine the leptin level or the eating pattern of the compound-treated mice. Normalization of the leptin level or eating pattern (e.g., the amount of food consumption) will indicate that the test compound can be used to treat obesity or certain eating disorders.
TIAR-deficient mice also have certain neurological disorders. For instance, the mice are unusually nervous. Using these mice, a skilled artisan can identify compounds useful in treating anxiety in humans.
Unless otherwise defined, 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. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice of the present invention. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
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Beck et al., “Structure, tissue distribution and genomic organization of the murine RRM-type . . . ”, Nucleic Acids Research 24:3829-3835, 1996.
Dember et al., “Individual RNA Recognition Motifs of TIA-1 and TIAR Have Different RNA . . . ”, The Journal of Biological Chemistry 271:2783-2788, 1996.
Kawakami et al., “Identification and functional characterization of a TIA-related nucleolysin”, Proc. Natl. Acad. Sci. USA 89:8681-8685, 1992.
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Tian et al., “A Polya
Anderson Paul
Beck Andreas
Streuli Michel
Dana-Farber Cancer Institute Inc.
Fish & Richardson P.C.
LeGuyader John L.
Wilson Michael C.
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