Attenuated protein expression vectors for use in siRNA rescue experiments.
Attenuated protein expression vectors for use in siRNA rescue experiments.
复制标题
DOI：
10.2144/000113909
复制
发表时间：
2012-08
期刊：
影响因子：
2.7
通讯作者：
Sundquist WI
中科院分区：
文献类型：
--
作者：
Morita E;Arii J;Christensen D;Votteler J;Sundquist WI
小干扰RNA （siRNA）的瞬时转染为研究细胞蛋白功能提供了一种强大的方法，特别是当目标蛋白可以从外源siRNA抗性构建体中重新表达以挽救敲低表型，确认siRNA靶点特异性并支持突变分析时。然而，当sirna抗性结构在次优水平表达时，救援实验经常失败。在这里，我们描述了一个具有不同强度CMV启动子的哺乳动物蛋白表达载体的集合。通过使用CHMP2A拯救HIV-1出芽，我们发现这些载体可以结合高转染效率和可调节的蛋白表达水平，以优化siRNA转染诱导的细胞表型的拯救。
Transient transfection of small interfering RNA (siRNA) provides a powerful approach for studying cellular protein functions, particularly when the target protein can be re-expressed from an exogenous siRNA-resistant construct in order to rescue the knockdown phenotype, confirm siRNA target specificity and support mutational analyses. Rescue experiments often fail, however, when siRNA-resistant constructs are expressed at suboptimal levels. Here, we describe an ensemble of mammalian protein expression vectors with CMV promoters of differing strengths. We show, using CHMP2A rescue of HIV-1 budding, that these vectors can combine high transfection efficiencies with tunable protein expression levels to optimize the rescue of cellular phenotypes induced by siRNA transfection.