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

隐性遗传筛选阐明胚胎干细胞中的 RNA 沉默

• 批准号: 7185336
• 负责人: XIAOZHONG ALEC WANG
• 金额: $ 18.88万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7185336
• 关键词: recessive; genetic; screen; elucidate; RNA

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.

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一个主要障碍是基因组的二倍体性质。为了克服这一障碍，我们一直使用布卢姆综合征蛋白缺陷 (blm-/-) 的 ES 细胞系。 Bloom缺陷细胞比野生型细胞表现出更高的杂合性损失，可用于产生筛选所需的纯合隐性突变体。利用 Bloom 缺陷型 ES 细胞的这一特征，我们设计了一种新型遗传筛选来鉴定哺乳动物 RNA 干扰 (RNAi) 途径的成分。 RNAi是最近发现的一种基因沉默现象，发生在多种生物体中。 RNAi 与多种生物和病理过程有关，从正常胚胎发育到人类癌症和神经退行性疾病。由于我们对 RNAi 途径的大部分了解来自对蠕虫、植物和苍蝇的遗传和生化分析，因此小鼠 ES 细胞中的遗传筛选将为研究脊椎动物系统中 RNAi 的独特方面提供令人兴奋的机会。在我们的设计中，RNAi 活性 blm-/- ES 细胞将使用随机整合到基因组内的逆转录病毒基因陷阱进行突变。 blm-/-细胞为逆转录病毒整合事件产生纯合突变体。然后我们将使用药物选择来分离假定的 RNAi 突变体。为了证明我们的设计原理，我们成功地进行了小规模筛选并鉴定了 Argonaute 2（RNAi 途径的已知组成部分）和其他假定的 RNAi 缺陷突变 ES 细胞系。因此，这些结果验证了我们对哺乳动物细胞中新型 RNAi 成分的筛选。我们建议使用我们在 Bloom 缺陷 ES 细胞中建立的选择系统对 RNAi 突变体进行大规模筛选。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检查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。