A novel recessive genetic screen for mitochondrial phenotypes in mammalian cells

描述（由申请人提供）：线粒体脑肌病是人类心血管疾病、智力低下和多系统疾病的重要病因。虽然线粒体疾病的个体原因很少，但最近的流行病学证据表明，呼吸链线粒体疾病的最低患病率为5000分之一。尽管最近对这些疾病的临床、生化和分子特征有了重要的见解，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然知之甚少，几乎没有有效的治疗方法。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，旨在识别对正常线粒体功能和线粒体疾病的潜在候选者都很重要的基因，从而为线粒体生物学和疾病提供新的见解。我们开发了一种使用高通量荧光激活细胞分选（FACS）来量化线粒体功能参数的方法，并使用逆转录病毒基因系统来鉴定影响线粒体功能的基因。我们已经证明，小鼠可以从携带已知导致人类线粒体疾病的基因突变的胚胎干细胞中产生。利用该系统，提出了一种在哺乳动物胚胎干细胞中进行隐性遗传筛选的新方法。一个传统的基因诱捕逆转录病毒载体已被修改，以包含一个诱导系统反义表达内源性诱捕位点。当被诱导时，预计产生的转录物会与内源性的感觉转录物结合，产生一个长双链RNA分子，激活内源性RNAi途径，并敲低完整的内源性位点的表达。此外，使用最近开发的全基因组shRNA慢病毒文库的RNAi筛选将被采用。这些不同方法的发展将代表一种创新手段，在胚胎干细胞中进行前所未有的隐性遗传筛选。突变小鼠ES细胞异常线粒体表型的鉴定为更好地理解正常线粒体功能、鉴定人类疾病的候选基因、为研究线粒体疾病的病理生理学和描绘潜在的治疗靶点产生新的动物模型提供了希望。

DESCRIPTION (provided by applicant): Mitochondrial encephalomyopathies are important causes of cardiovascular disease, mental retardation and multisystem disease in humans. While individual causes of mitochondrial disease are rare, recent epidemiological evidence suggests that the minimal prevalence of respiratory chain mitochondrial diseases is 1 in 5000. Despite important recent 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, with virtually no effective therapies available. The overall goal of this project is to provide new insights into mitochondrial biology and disease by performing a genetic screen in mouse embryonic stem (ES) cells that is designed to identify genes that are important for both normal mitochondrial function and are potential candidates for mitochondrial diseases. We have developed a means to use high throughput fluorescence activated cell sorting (FACS) to quantify parameters of mitochondrial function and have used a retroviral gene system to identify genes affecting mitochondrial function. We have demonstrated that mice can be generated from ES cells that carry mutations in genes known to cause human mitochondrial disease. Using this system, a novel method to perform a recessive genetic screen in mammalian ES cells is proposed. A traditional gene trap retroviral vector has been modified to contain an inducible system for antisense expression of the endogenous trapped locus. When induced, the resulting transcript is predicted to bind to the endogenous sense transcript, resulting in a long, double stranded RNA molecule that will activate endogenous RNAi pathways and will knockdown expression of the intact endogenous locus. In addition, an alternative RNAi screen using recently developed genome-wide shRNA lentivirus library will be employed. The development of these different approaches would represent an innovative means to perform unprecedented recessive genetic screens in ES cells. The identification of abnormal mitochondrial phenotypes in mutagenized mouse ES cells offers the promise of better understnading normal mitochondrial function, identifying candidate genes for human disease, generating new animal models for studying the pathophysiology of mitochondrial disease and delineating potential therapeutic targets. PUBLIC HEALTH RELEVANCE: Mitochondria are the structures found in cells that generate energy and regulate cell death. This proposal is to generate cell lines and animal models where specific genes that have some mitochondria functions are disrupted by novel genetic research tools.