Genetic Screens for Mitochondrial Phenotypes in ES Cells

描述(申请人提供)：线粒体肌病是骨骼肌疾病的一个重要原因，也是许多线粒体疾病的共同临床特征。最近的流行病学证据表明，线粒体疾病的患病率可能高达8500人中有1人。尽管对这些疾病的临床、生化和分子特征有重要的见解，但只有少数病例确定了特定的遗传病因，其潜在的分子发病机制仍然知之甚少。该项目的总体目标是通过在小鼠胚胎干细胞(ES)中进行基因筛选来识别对线粒体功能重要的基因以及可能导致线粒体肌病的基因，从而为线粒体生物学提供新的见解。经启动子-陷阱策略突变的ES细胞显示出异常的线粒体表型，将通过使用线粒体质量和线粒体膜电位的荧光标记物的荧光激活细胞分选(FACS)来选择。FACS显示线粒体质量和/或线粒体膜电位可重复性改变的克隆将通过5‘RACE进行分析，以鉴定改变的基因。突变克隆将进一步在生化、形态和超微结构水平上进行特征分析。遗传特异性将通过表型挽救由候选基因的cDNA转染的突变克隆来证明。通过结合小鼠ES细胞的遗传易操纵性和FACS的高通量表型能力，该建议提供了一种新的、快速的方法来识别哺乳动物系统中对线粒体功能重要的基因，该系统非常适合研究肌肉生物学。进行这项研究的环境非常适合支持申请人发展线粒体肌病和生物学的研究计划。贝勒医学院以其哺乳动物遗传学而闻名，并提供访问最先进的FACS核心的途径。

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.