Genetic Screens for Mitochondrial Phenotypes in ES Cells

ES 细胞线粒体表型的遗传筛选

• 批准号: 6867240
• 负责人: Brett Harrison Graham
• 金额: $ 7.5万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-17 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6867240
• 关键词: biotechnology; embryonic stem cell; flow cytometry; fluorescent dye /probe; genetic screening; high throughput technology; membrane potentials; mitochondrial DNA; mitochondrial disease /disorder; phenotype; tissue /cell culture; transfection

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.

描述（由申请人提供）：线粒体肌病是骨骼肌疾病的重要原因，并且是许多线粒体疾病的常见临床特征。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 分之一。尽管对这些疾病的临床、生化和分子特征有了重要的了解，但仅在少数病例中确定了特定的遗传病因，并且潜在的分子发病机制仍知之甚少。该项目的总体目标是通过在小鼠胚胎干 (ES) 细胞中进行遗传筛选，旨在识别对线粒体功能重要的基因以及线粒体肌病可能的病因候选基因，从而为线粒体生物学提供新的见解。通过启动子捕获策略诱变的显示异常线粒体表型的 ES 细胞将通过荧光激活细胞分选 (FACS) 使用线粒体质量和线粒体膜电位的荧光标记进行选择。 FACS 表明线粒体质量和/或线粒体膜电位发生可重现改变的克隆将通过 5' RACE 进行分析，以鉴定改变的基因。然后，突变克隆将在生化、形态学和超微结构水平上得到进一步表征。遗传特异性将通过候选基因 cDNA 转染的突变克隆的表型拯救来证明。通过将小鼠 ES 细胞的遗传易处理性与 FACS 的高通量表型分析能力相结合，该提案提供了一种新颖、快速的方法，用于在非常适合研究肌肉生物学的哺乳动物系统中鉴定对线粒体功能重要的基因。进行这项研究的环境非常适合支持申请人开发线粒体肌病和生物学研究项目。贝勒医学院以其哺乳动物遗传学而闻名，并提供最先进的 FACS 核心。