Trombly Melanie I, Wang Xiaozhong
Department of Biochemistry, Northwestern University, Evanston, IL, USA.
Methods Mol Biol. 2010;650:45-63. doi: 10.1007/978-1-60761-769-3_4.
Several key components of the RNA interference (RNAi) pathway were identified in genetic screens performed in nonmammalian model organisms. To identify components of the mammalian RNAi pathway, we developed a recessive genetic screen in mouse embryonic stem (ES) cells. Recessive genetic screens are feasible in ES cells that are Bloom-syndrome protein (Blm-) deficient. Therefore, we constructed a reporter cell line in Blm-deficient ES cells to isolate RNAi mutants through a simple drug-selection scheme. This chapter describes how we used retroviral gene traps to mutagenize the reporter cell line and select for RNAi mutants. Putative RNAi mutants were confirmed using a separate functional assay. The location of the gene trap was then identified using molecular techniques such as Splinkerette PCR. Our screening strategy successfully isolated several mutant clones of Argonaute2, a vital component of the RNAi pathway.
在非哺乳动物模式生物中进行的遗传筛选中，鉴定出了RNA干扰（RNAi）途径的几个关键组成部分。为了鉴定哺乳动物RNAi途径的组成部分，我们在小鼠胚胎干细胞（ES细胞）中开发了一种隐性遗传筛选方法。隐性遗传筛选在缺乏布鲁姆综合征蛋白（Blm-）的ES细胞中是可行的。因此，我们在Blm缺陷的ES细胞中构建了一个报告细胞系，通过简单的药物选择方案分离RNAi突变体。本章描述了我们如何使用逆转录病毒基因陷阱诱变报告细胞系并筛选RNAi突变体。使用单独的功能测定法确认推定的RNAi突变体。然后使用诸如Splinkerette PCR等分子技术鉴定基因陷阱的位置。我们的筛选策略成功分离出了RNAi途径的重要组成部分AGO2的几个突变克隆。
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