ALTERED LUNG GLUTATHIONE INDUCED--INSERTION MUTAGENESIS

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Toxic oxygen species are important mediators of lung injury. An array of antioxidant defenses have evolved to protect cells against reactive oxygen species. An important component of this defensive system is glutathione. The specific aims in this study are to identify and characterize genetic factors which can influence the intracellular concentration of glutathione in mouse embryonic stem cells, and to generate transgenic mice from these cells. We hypothesize that selected mouse embryonic stems cells with alterations in glutathione content will display either increased or decreased resistance to reactive oxygen metabolites. Transgenic mice formed from such stem cells should also show altered sensitivity to free-radical damage. During the study period we will 1) isolate retrovirus-induced mutants of mouse embryonic stem cells with altered GSH content, by using a fluorescent dye specific for GSH; 2) isolate and characterize the mutated DNA that resulted in altered GSH metabolism; 3) analyse the effects of the altered GSH on cell sensitivity to free-radical damage; and 4) establish colonies of transgenic mice bearing the GSH mutations of interest. Creation of transgenic mice with genetically altered GSH content will be especially useful in future studies of free radical-induced lung injury.