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Christopher P Austin,
James F Battey,
Allan Bradley,
Maja Bucan,
Mario Capecchi,
Francis S Collins,
William F Dove,
Geoffrey Duyk,
Susan Dymecki,
Janan T Eppig, [......],
Lyuba Varticovski,
Inder M Verma,
Thomas F Vogt,
Harald von Melchner,
Jan Witkowski, Richard P Woychik,
Wolfgang Wurst,
George D Yancopoulos,
Stephen G Young,
Brian Zambrowicz
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ABSTRACT: Mouse knockout technology provides a powerful means of elucidating gene function in vivo, and a publicly available genome-wide collection of mouse knockouts would be significantly enabling for biomedical discovery. To date, published knockouts exist for only about 10% of mouse genes. Furthermore, many of these are limited in utility because they have not been made or phenotyped in standardized ways, and many are not freely available to researchers. It is time to harness new technologies and efficiencies of production to mount a high-throughput international effort to produce and phenotype knockouts for all mouse genes, and place these resources into the public domain.
Nature Genetics 10/2004; 36(9):921-4. · 35.53 Impact Factor
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ABSTRACT: The agouti protein is a paracrine factor that is normally present in the skin of many species of mammals. Agouti regulates the switch between black and yellow hair pigmentation by signalling through the melanocortin 1 receptor (Mc1r) on melanocytes. Lethal yellow (Ay) and viable yellow (Avy) are dominant regulatory mutations in the mouse agouti gene that cause the wild-type protein to be produced at abnormally high levels throughout the body. Mice harboring these mutations exhibit a pleiotropic syndrome characterized by yellow coat color, obesity, hyperglycemia, hyperinsulinemia, and increased susceptibility to hyperplasia and carcinogenesis in numerous tissues, including the liver. The goal of this research was to determine if ectopic expression of the agouti gene in the liver alone is sufficient to recapitulate any aspect of this syndrome. For this purpose, we generated lines of transgenic mice expressing high levels of agouti in the liver under the regulatory control of the albumin promoter. Expression levels of the agouti transgene in the liver were quantified by Northern blot analysis. Functional agouti protein in the liver of transgenic mice was assayed by its ability to inhibit binding of the alpha-melanocyte stimulating hormone (alphaMSH) to the Mc1r. Body weight, plasma insulin and blood glucose levels were analyzed in control and transgenic mice. Control and transgenic male mice were given a single intraperitoneal injection (10 mg/kg) of the hepatocellular carcinogen, diethylnitrosamine (DEN), at 15 days of age. Mice were euthanized at 36 or 40 weeks after DEN injection and the number of tumors per liver and total liver weights were recorded.
The albumin-agouti transgene was expressed at high levels in the livers of mice and produced a functional agouti protein. Albumin-agouti transgenic mice had normal body weights and normal levels of blood glucose and plasma insulin, but responded to chemical initiation of the liver with an increased number of liver tumors compared to non-transgenic control mice.
The data demonstrate that liver-specific expression of the agouti gene is not sufficient to induce obesity or diabetes, but, in the absence of these factors, agouti continues to promote hepatocellular carcinogenesis.
Molecular Cancer 07/2004; 3:17. · 3.99 Impact Factor
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ABSTRACT: Tg737 mutant mice exhibit pathologic conditions in numerous tissues along with skeletal patterning defects. Herein, we characterize the skeletal pathologic conditions and confirm a role for Tg737 in skeletal patterning through transgenic rescue. Analyses were conducted in both the hypomorphic Tg737(orpk) allele that results in duplication of digit one and in the null Tg737(delta2-3betaGal) allele that is an embryonic lethal mutation exhibiting eight digits per limb. In early limb buds, Tg737 expression is detected throughout the mesenchyme becoming concentrated in precartilage condensations at later stages. In situ analyses indicate that the Tg737(orpk) mutant limb defects are not associated with changes in expression of Shh, Ihh, HoxD11-13, Patched, BMPs, or Glis. Likewise, in Tg737(delta2-3betaGal) mutant embryos, there was no change in Shh expression. However, in both alleles, Fgf4 was ectopically expressed on the anterior apical ectodermal ridge. Collectively, the data argue for a dosage effect of Tg737 on the limb phenotypes and that the polydactyly is independent of Shh misexpression.
Developmental Dynamics 06/2003; 227(1):78-90. · 2.54 Impact Factor
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ABSTRACT: Agouti is a paracrine-acting, transient antagonist of melanocortin 1 receptors that specifies the subapical band of yellow on otherwise black hairs of the wild-type coat. To better understand both agouti structure/function and the germline damage caused by chemicals and radiation, an allelic series of 25 recessive, homozygous-viable agouti mutations generated in specific-locus tests were characterized. Visual inspection of fur, augmented by quantifiable chemical analysis of hair melanins, suggested four phenotypic categories (mild, moderate, umbrous-like, severe) for the 18 hypomorphs and a single category for the 7 amorphs (null). Molecular analysis indicated protein-coding alterations in 8 hypomorphs and 6 amorphs, with mild-moderate phenotypes correlating with signal peptide or basic domain mutations, and more devastating phenotypes resulting from C-terminal lesions. Ten hypomorphs and one null demonstrated wild-type coding potential, suggesting that they contain mutations elsewhere in the > or = 125-kb agouti locus that either reduce the level or alter the temporal/spatial distribution of agouti transcripts. Beyond the notable contributions to the field of mouse germ cell mutagenesis, analysis of this allelic series illustrates that complete abrogation of agouti function in vivo occurs most often through protein-coding lesions, whereas partial loss of function occurs slightly more frequently at the level of gene expression control.
Genetics 02/2002; 160(2):659-74. · 4.01 Impact Factor
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ABSTRACT: We have developed a new procedure utilizing microhomologous recombination in yeast to generate targeting constructs for producing targeted mutations in mice. This procedure is rapid and efficient, and should be directly applicable to all mammalian genes. Moreover, only minimal information about the locus being targeted is required. The feasibility of this approach was demonstrated by producing another allele of the mouse Tg737 polycystic kidney gene.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 07/1998; · 2.85 Impact Factor
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ABSTRACT: A human homolog (RALY) of the mouse Raly gene was isolated and sequenced, and shown to encode a novel protein isoform containing a 16 amino acid in-frame insert in the variable region of the protein. Analysis of the corresponding region of the mouse Raly gene demonstrated that this novel protein isoform is also present in the mouse. Comparative analysis of RALY cDNA and EST sequences suggests the presence of additional alternatively processed RALY transcripts. As in the mouse, the human RALY gene is widely expressed as a 1.7-kb transcript.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression.
