A Recessive Genetic Screen for Components of the RNA Interference Pathway Performed in Mouse Embryonic Stem Cells.
A Recessive Genetic Screen for Components of the RNA Interference Pathway Performed in Mouse Embryonic Stem Cells.
复制标题
在小鼠胚胎干细胞中进行 RNA 干扰途径成分的隐性遗传筛选。
DOI：
10.1007/978-1-4939-7108-4_9
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发表时间：
2017
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鉴定了RNA干扰（RNAi）途径的几个关键组分。为了鉴定哺乳动物RNAi途径的组分，我们在小鼠胚胎干细胞（ES）中开发了隐性遗传筛选。隐性遗传筛选在布卢姆综合征蛋白缺陷（Blm缺陷）的ES细胞中是可行的。我们在Blm缺陷的ES细胞中构建了一个报告细胞系，使用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和选择RNAi突变体。使用单独的功能测定法确认推定的RNAi突变体。然后使用分子技术如splinkerette PCR鉴定基因陷阱的位置。我们的筛选策略成功地分离了Argonaute 2的几个突变克隆，Argonaute 2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 non-mammalian 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 deficient (Blm-deficient). We constructed a reporter cell line in Blm-deficient ES cells to isolate RNAi mutants using 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 Argonaute 2, a vital component of the RNAi pathway.