Genetic Screens for Mitochondrial Phenotypes in ES Cells

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DESCRIPTION (provided by applicant): Mitochondrial myopathy is an important cause of skeletal muscle disease and is a common clinical feature of many mitochondrial disorders. Recent epidemiological evidence suggests that the prevalence of mitochondrial diseases may be as high as 1 in 8500. Despite important insights into the clinical, biochemical, and molecular characterization of these disorders, specific genetic etiologies have been identified in only a minority of cases, and the underlying molecular pathogenesis remains poorly understood. The overall goal of this project is to provide new insights into mitochondrial biology by performing genetic screens in mouse embryonic stem (ES) cells designed to identify genes that are important for mitochondrial function and that are possible etiological candidates for mitochondrial myopathies. ES cells mutagenized by a promoter-trap strategy that demonstrate an abnormal mitochondrial phenotype will be selected by fluorescence-activated cell sorting (FACS) using fluorescent markers for mitochondrial mass and mitochondrial membrane potential. Clones with reproducible altered mitochondrial mass and/or mitochondrial membrane potential as indicated by FACS will then be analyzed by 5' RACE to identify the altered gene. Mutant clones will then be further characterized on biochemical, morphological, and ultrastructural levels. Genetic specificity will be demonstrated by phenotypic rescue of mutant clones transfected by the cDNA of the candidate gene. By combining the genetic tractability of mouse ES cells with the high-throughput phenotyping capacity of FACS, this proposal offers a novel, rapid approach for identifying genes important for mitochondrial function in a mammalian system well-suited for studying muscle biology. The environment in which this study will be performed is uniquely suited for supporting the applicant's development of a research program in mitochondrial myopathy and biology. Baylor College of Medicine is renowned for its mammalian genetics and provides access to a state of the art FACS core.