Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
2002-02-28
2004-07-13
Paras, Peter (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C435S325000
Reexamination Certificate
active
06762343
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention is directed towards specific double knockout(DKO) animals and their use as animal models. More specifically, the double knockout animals contain a disruption in the genes encoding GPX-1 and GPX-GI. Corresponding cells which are amenable to tissue culture are also part of the invention, as are methods of using such cells, including their use as a tool for identifying therapeutic agents. In addition, the invention is directed towards a mouse model of cancer of the small bowel.
BACKGROUND OF THE INVENTION
Selenium-dependent glutathione peroxidases (GPXs) are a family of enzymes that are most efficient in the reduction of hydroperoxides. At the present time there are four known selenoproteins with GPX activity. These include the classical ubiquitous GPX-1, GPX-GI, the secreted GPX-P, and PHGPX (Ursini, F., Heim, S., Kiess, M., Maiorino, M., Roveri, A., Wissing, J. & Flohe, L. (1999) Science 285, 1393-6). These GPX isozymes are encoded by four distinct genes, the Gpx1 gene for GPX-1, the Gpx2 gene for GPX-GI, the Gpx3 gene for GPX-P, and the Gpx4 gene for PHGPX (Chu, F. F. (1994) Cytogenet Cell Genet 66, 96-8). The GPX-1 and GPX-GI isozymes have very similar properties including substrate specificity and cytosolic localization (Chu, F. F., Doroshow, J. H. & Esworthy, R. S. (1993) J. Biol Chem 268, 2571-6; Esworthy, R. S., Swiderek, K. M., Ho, Y. S. & Chu, F. F. (1998) Biochim Biophys Acta 1381,213-26). The unique feature of GPX-GI is its high levels of expression in the epithelium of the GI-tract. GPX-P is found in body fluids such as plasma, lung and GI-tract (Tham, D. M., Whitin, J. C., Kim, K. K., Zhu, S. X. & Cohen, H. J. (1998) Am J Physiol 275, G1463-71; Kim, K. K., Whitin, J. C., Sukhova, N. M. & Cohen, H. J. (1999) Pediatr Res 46, 715-21). PHGPX is present at a high level in testis and is implicated in sperm maturation (1, Roveri, A., Casasco, A., Maiorino, M., Dalan, P., Cafligaro, A. & Ursini, F. (1992) J Biol Chem 267, 6142-6). PHGPX is present in low level in the GI-tract (Chu, F. F. & Esworthy, R. S. (1995) Arch. Biochem. Biophys. 323, 288-94). Both GPX-P and PHGPX can reduce phospholipid and cholesterol hydroperoxides, while the latter is a more efficient enzyme at reducing these substrates (Thomas, J. P., Maiorino, M., Ursini, F. & Girotti, A. W. (1990) J. Biol. Chem. 265, 454-6 1; Esworthy, R. S., Chu, F. F., Geiger, P., Girotti, A. W. & Doroshow, J. H. (1993) Arch. Biochem. Biophys. 307, 29-34).
It is known that knockout (KO) mice homozygous for disruption of single Gpx1 and Gpx2 genes (i.e., Gpx1-KO and Gpx2-KO mice) display little pathology without additional stress (Ho, Y. S., Magnenat, J. L., Bronson, R. T., Cao, J., Gargano, M., Sugawara, M. & Funk, C. D. (1997) J. Biol. Chem. 272, 16644-51; Esposito, L. A., Kokoszka, J. E., Waymire, K. G., Cottrell, B., MacGregor, G. R. & Wallace, D.C. (2000) Free Radic. Biol. Med. 28, 754-66; Esworthy, R. S., Mann, J. R., Sam, M. & Chu, F. (2000) Am. J. Physiol. Gastrointest. Liver Physiol. 279, G426-G436.). However, aged Gpx1-KO mice display spontaneous weight loss (Esposito, L. A., Kokoszka, J. E., Waymire, K. G., Cottrell, B., MacGregor, G. R. & Wallace, D.C. (2000) Free Radic. Biol. Med. 28, 754-66). For example, the Gpx1 gene is expressed ubiquitously and is highly expressed in the erythrocyte, liver, and kidney. The antioxidant function of GPX-1 is revealed in Gpx1-KO mice especially after treatment with prooxidant chemicals. An increased level of H
2
O
2
is produced from liver mitochondria in older (5-6 month) Gpx1-KO mice compared with that from wildtype mice without any treatment. GPX-1 can prevent lipid peroxidation induced by paraquat herbicide or measured in liver and lung and protect cortical neurons against H
2
O
2
(Cheng, W. H., Ho, Y. S., Valentine, B. A., Ross, D. A., Combs, G. F., Jr. & Lei, X. G. (1998) J. Nutr. 128,1070-6; de Haan, J. B., Bladier, C., Griffiths, P., Kelner, M., O'Shea, R. D., Cheung, N. S., Bronson, R. T., Silvestro, M. J., Wild, S., Zheng, S. S., Beart, P. M., Hertzog, P. J. & Kola, 1. (1998) J. Biol. Chem. 273, 22528-36). The Gpx1-KO mice are more susceptible to neurotoxic agents as malonate, 3-nitropropionic acid, and 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) in the brain (Klivenyi, P., Andreassen, O. A., Ferrante, R. J., Dedeoglu, A., Mueller, G., Lancelot, E., Bogdanov, M., Andersen, J. K., Jiang, D. & Beal, M. F. (2000) J. Neurosci. 20, 1-7). Malonate induces hydroxyl radical generation while MPP+, the active metabolite of MPTP, inhibits mitochondrial complex I activity (Tipton, K. F. & Singer, T. P. (1993) J Neurochem 61, 1191-206). Since these neurotoxins produce oxidative stress and impair energy production, GPX-1 is implicated in protection against oxidative damage.
Depending on the type of insult, lack of GPX activity can also be beneficial under certain circumstances. Mice overexpressing GPX-1 and GPX-P have low levels of peroxides and prostaglandins and are more sensitive to hyperthermia (Mirochnitchenko, O., Palnitkar, U., Philbert, M. & Inouye, M. (1995) Proc. Natl. Acad. Sci. USA 92, 8120-4). We have found that the jejunum crypt of Gpx1-KO mice regenerates better than that in the wildtype after exposure to high dose &ggr;-irradiation (Esworthy, R. S., Mann, J. R., Sam, M. & Chu, F. (2000) Am J Physiol Gastrointest Liver Physiol 279, G426). Perhaps the higher level of GPX-GI in the Gpx1-KO mouse intestine are directly responsible for crypt regeneration. Furthermore, the lack of GPX activity in the Gpx1-KO mouse can be protective against kainic acid-induced limbic seizures and neurodegeneration (Jiang, D., Akopian, G., Ho, Y. S., Walsh, J. P. & Andersen, J. K. (2000) Exp. Neurol. 164, 257-68). This appears to result from decreased receptor function for N-methyl-D-aspartate (NMDA), since kainic acid induces NMDA-dependent seizure. Increased GPX activity in mice overexpressing the Gpx1 gene may have enhanced carcinogenic response in skin treated with 7,12-dimethylbenz[a]anthracene and 2-0-tetradecanoylphorbol-13-acetate (Lu, Y. P., Lou, Y. R., Yen, P., Newmark, H. L., Nfirochnitchenko, 0. I., Inouye, M. & Huang, M. T. (1997) Cancer Res. 57, 1468-74). The mechanism of the pro-carcinogenic activity is not known but it is apparent that elevated antioxidant activity can be debilitating, depending on the type of insult.
GPX activity is also implicated in protection against infectious agents. For example, Jaeschke et al. have found that Gpx1-KO mice are more susceptible to neutrophil-mediated parenchymal cell injury during endotoxemia (Jaeschke, H., Ho, Y. S., Fisher, M. A., Lawson, J. A. & Farhood, A. (1999) Hepatology 29,443-50). The galactosamine/endotoxin induced acute liver failure involves neutrophils and GPX protects hepatocytes against peroxides generated by infiltrated neutrophils in the liver. It has also previously been shown that Gpx1-KO mice are more susceptible to coxsackievirus-induced myocarditis (Beck, M. A., Esworthy, R. S., Ho, Y. S. & Chu, F. F. (1998) Faseb J. 12,1143-9). Viral antibody titers in the Gpx1-KO mice are less than 20% of those found in the wildtype mice, suggesting that cellular immune response is impaired in the Gpx1-KO mice.
Gpx2-KO mice have also recently been generated and these mice appear to be normal (Esworthy et al., Am. J. Physiol. Gastrointest. Liver Physiol. 279, G426-G436). Unlike the Gpx1 gene, which is expressed ubiquitously, the Gpx2 gene is expressed specifically in epithelium. The Gpx2 gene is highly expressed in the gastrointestinal tract, and is also present in the breast, lung, and human liver (Chu, F. F., Doroshow, J. H. & Esworthy, R. S. (1993) J. Biol. Chem. 268, 2571-6; Chu, F. F., Esworthy, R. S., Lee, L. & Wilczynski, S. (1999) J. Nutr. 129, 1846-1854). In the GI-epithelium, GPX-1 and GPX-GI contribute to most of GPX activity (Esworthy, R. S., Swiderek, K. M., Ho, Y. S. & Chu, F. F. (1998) Biochim. Biophys. Acta. 1381,213-26.). The lack of pathology in Gpx2-KO mice is not unexpected, since the Gpx2 gene has limited tissue expression, the
Chu Fong-Fong
Doroshow James H.
Esworthy Robert S.
City of Hope
Paras Peter
Rothwell Figg Ernst & Manbeck
LandOfFree
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