A recessive genetic screen to elucidate RNA silencing in embryonic stem cells

描述（由申请人提供）：胚胎干（ES）细胞可用于广泛的遗传操作，因此，提供了一种揭示人类基因功能的方法。我们的实验室致力于建立使用ES细胞进行遗传分析的新方法。在哺乳动物细胞中进行隐性遗传筛选的主要障碍是基因组的二倍体性质。为了克服这一障碍，我们一直在使用布卢姆综合征蛋白缺陷（blm-/-）的ES细胞系。布卢姆缺陷细胞表现出比野生型细胞高得多的杂合性丢失，并且可用于产生筛选所需的纯合隐性突变体。利用Bloom缺陷ES细胞的这一特点，我们设计了一种新的遗传筛选来鉴定哺乳动物RNA干扰（RNAi）途径的组分。RNAi是近年来发现的一种基因沉默现象，广泛存在于多种生物体中。RNAi参与了从正常胚胎发育到人类癌症和神经退行性疾病的多种生物学和病理学过程。由于我们对RNAi途径的理解大多来自于蠕虫、植物和苍蝇的遗传和生化分析，因此在小鼠ES细胞中进行遗传筛选将为研究RNAi在脊椎动物系统中的独特方面提供令人兴奋的机会。在我们的设计中，RNAi感受态blm-/- ES细胞将使用随机整合在基因组内的逆转录病毒基因陷阱进行突变。blm-/-细胞产生逆转录病毒整合事件的纯合突变体。然后，我们将使用药物选择来分离推定的RNAi突变体。为了证明我们的设计原理，我们已经成功地进行了小规模的筛选，并确定Argonaute 2，一个已知的RNAi途径的组成部分和其他推定的RNAi缺陷突变体ES细胞系。因此，这些结果验证了我们在哺乳动物细胞中筛选新的RNAi组分。我们建议在Bloom缺陷的ES细胞中使用我们建立的选择系统进行大规模的RNAi突变体筛选。通过在哺乳动物细胞中完成全基因组隐性遗传筛选，我们不仅将有独特的机会在脊椎动物基因组的未探索领域中研究RNAi途径，而且还为使用隐性遗传筛选破译其他遗传途径树立了榜样。

DESCRIPTION (provided by applicant): Embryonic stem (ES) cells are accessible for extensive genetic manipulations, therefore, providing a means to unveil functions of human genes. Our lab focuses on establishing new approaches for genetic analysis using ES cells. A major obstacle to performing recessive genetic screens in mammalian cells is the diploid nature of the genome. To overcome this barrier, we have been using an ES cell line that are Bloom- syndrome protein deficient (blm-/-). Bloom-deficient cells exhibit a much higher loss of heterozygosity than wild-type cells and can be used to generate homozygous recessive mutants required for screening. Taking advantage of this feature of Bloom deficient ES cells, we have devised a novel genetic screen to identify components of mammalian RNA interference (RNAi) pathway. RNAi is a recently discovered gene silencing phenomenon that occurs in a wide variety of organisms. RNAi has been implicated in a variety of biological and pathological processes, from normal embryonic developemnt to human cancers and neurodegenerative diseases. Because much of our understanding of RNAi pathway come from genetic and biochemical analysis in worms, plants and flies, a genetic screen in mouse ES cells will provide an exciting opportunity to examine the unique aspect of RNAi in a vertebrate system. In our design, RNAi competent blm-/- ES cells will be mutated using a retroviral gene trap that randomly integrates within the genome. The blm-/- cells create homozygous mutants for the retroviral integration events. We will then use drug selection to isolate putative RNAi mutants. To prove the principle of our design, we have successfully performed a small-scale screen and identified Argonaute 2, a known component of RNAi pathway and other putative RNAi defective mutant ES cell lines. These results have therefore validated our screen for novel RNAi components in mammalian cells. We propose to carry out a large scale screen for RNAi mutants using our established selection system in Bloom deficient ES cells. By completing a genome-wide recessive genetic screen in mammalian cells, we will not only have the unique opportunity to examine the RNAi pathway in the unexplored territory of the vertebrate genome, but also set an example for deciphering other genetic pathways using recessive genetic screens.