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、精神发育迟滞和多系统疾病的重要原因。虽然线粒体疾病的个别原因是罕见的，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1/5000。尽管最近对这些疾病的临床、生化和分子特征有了重要的认识，但仅在少数病例中确定了特定的遗传病因，并且对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，为线粒体生物学和疾病提供新的见解，该细胞旨在识别对正常线粒体功能重要的基因，并且是线粒体疾病的潜在候选者。我们已经开发了一种方法，使用高通量荧光激活细胞分选（FACS）来量化线粒体功能的参数，并使用逆转录病毒基因系统来识别影响线粒体功能的基因。我们已经证明，小鼠可以从ES细胞产生，这些细胞携带已知引起人类线粒体疾病的基因突变。利用该系统，提出了一种在哺乳动物ES细胞中进行隐性遗传筛选的新方法。传统的基因诱捕逆转录病毒载体已被修改为包含用于内源性诱捕基因座的反义表达的诱导型系统。当被诱导时，预测所得转录物与内源有义转录物结合，产生长的双链RNA分子，其将激活内源RNAi途径并将敲低完整内源基因座的表达。此外，还将使用最近开发的全基因组shRNA慢病毒文库进行另一种RNAi筛选。这些不同方法的发展将代表在ES细胞中进行前所未有的隐性遗传筛选的创新手段。突变小鼠ES细胞中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、产生用于研究线粒体疾病的病理生理学的新动物模型以及描绘潜在的治疗靶点提供了希望。 公共卫生相关性：线粒体是细胞中发现的产生能量和调节细胞死亡的结构。该提案旨在产生细胞系和动物模型，其中具有某些线粒体功能的特定基因被新的遗传研究工具破坏。

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.