A recessive genetic screen for components of the RNA interference pathway in mouse embryonic stem cells.
A recessive genetic screen for components of the RNA interference pathway in mouse embryonic stem cells.
复制标题
对小鼠胚胎干细胞中 RNA 干扰途径成分的隐性遗传筛选。
DOI：
10.1007/978-1-60761-769-3_4
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
Wang,Xiaozhong
中科院分区：
文献类型：
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作者：
Trombly,MelanieI;Wang,Xiaozhong
在非哺乳动物模式生物中进行的遗传筛选中，确定了RNA干扰(RNAi)途径的几个关键成分。为了确定哺乳动物RNAi途径的组成部分，我们在小鼠胚胎干细胞中开发了一种隐性遗传筛选。隐性遗传筛查在缺乏Bloom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细胞中构建了报告细胞系，通过一种简单的药物选择方案来分离RNAi突变体。本章描述了我们如何使用逆转录病毒基因陷阱来诱变报告细胞系，并选择RNAi突变体。使用单独的功能分析确认了可能的RNAi突变体。然后使用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地分离了ArgAerte2的几个突变克隆，ArgAerte2是RNAi途径的重要组成部分。
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.