Morita Eiji, Arii Jun, Christensen Devin, Votteler Jörg, Sundquist Wesley I
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT, USA.
Biotechniques. 2012 Aug;0(0):1-5. doi: 10.2144/000113909.
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. Using CHMP2A rescue of HIV-1 budding, we show that these vectors can combine high-transfection efficiencies with tunable protein expression levels to optimize the rescue of cellular phenotypes induced by siRNA transfection.
小干扰RNA（siRNA）的瞬时转染为研究细胞蛋白质功能提供了一种强大的方法，特别是当靶蛋白可以从外源性抗siRNA构建体中重新表达，以挽救敲低表型、确认siRNA靶标特异性并支持突变分析时。然而，当抗siRNA构建体以次优水平表达时，挽救实验往往会失败。在这里，我们描述了一组具有不同强度CMV启动子的哺乳动物蛋白质表达载体。通过使用CHMP2A挽救HIV-1出芽，我们表明这些载体可以将高转染效率与可调节的蛋白质表达水平相结合，以优化由siRNA转染诱导的细胞表型的挽救。
