An inducible system for expression and validation of the specificity of short hairpin RNA in mammalian cells.
An inducible system for expression and validation of the specificity of short hairpin RNA in mammalian cells.
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DOI：
10.1093/nar/gkl1109
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发表时间：
2007
影响因子：
14.9
通讯作者：
Poon, Randy Y. C.
中科院分区：
文献类型：
--
作者：
Ma, Hoi Tang;On, Kin Fan;Tsang, Yiu Huen;Poon, Randy Y. C.
短发夹RNA （short hairpin RNA, shRNA）介导的RNA干扰（RNA interference, RNAi）已成为分析哺乳动物细胞功能丧失的有力工具。RNAi实验的主要问题是脱靶效应，而最有力地证明shRNA特异性的是用耐shRNA的靶基因来挽救RNAi效应。这带来了自身的问题，包括shRNA和拯救cDNA在单个细胞中的相对表达不可预测，以及难以产生稳定的细胞系。在本报告中，我们评估了shRNA和救援cDNA在同一载体上联合表达的可行性。除了便于验证shRNA特异性外，该系统还大大简化了shRNA表达细胞系的生成。由于补偿性cDNA受诱导启动子的控制，在研究敲低表型之前，通过有条件地关闭拯救蛋白，可以产生稳定的shrna表达细胞。相反，在内源蛋白被完全抑制后，救援蛋白可以被激活。当shRNA或补偿性cDNA的长时间表达对细胞生长有害时，这种方法特别适用。该系统可以方便地一步验证shRNA和生成稳定的shRNA表达细胞。
RNA interference (RNAi) by means of short hairpin RNA (shRNA) has developed into a powerful tool for loss-of-function analysis in mammalian cells. The principal problem in RNAi experiments is off-target effects, and the most vigorous demonstration of the specificity of shRNA is the rescue of the RNAi effects with a shRNA-resistant target gene. This presents its own problems, including the unpredictable relative expression of shRNA and rescue cDNA in individual cells, and the difficulty in generating stable cell lines. In this report, we evaluated the plausibility of combining the expression of shRNA and rescue cDNA in the same vector. In addition to facilitate the validation of shRNA specificity, this system also considerably simplifies the generation of shRNA-expressing cell lines. Since the compensatory cDNA is under the control of an inducible promoter, stable shRNA-expressing cells can be generated before the knockdown phenotypes are studied by conditionally turning off the rescue protein. Conversely, the rescue protein can be activated after the endogenous protein is completely repressed. This approach is particularly suitable when prolonged expression of either the shRNA or the compensatory cDNA is detrimental to cell growth. This system allows a convenient one-step validation of shRNA and generation of stable shRNA-expressing cells.
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通讯作者：
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