Kanungo Jyotshna
Division of Neurotoxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, 3900 NCTR Road, Jefferson, AR 72079, USA.
Biochem Biophys Res Commun. 2017 Apr 29;486(2):481-485. doi: 10.1016/j.bbrc.2017.03.066. Epub 2017 Mar 16.
RNA silencing is used as a common method for investigating loss-of-function effects of genes of interest. In mammalian cells, RNA interference (RNAi) or RNA silencing can be achieved by transient siRNA (small or short interfering RNA) transfection or by stable shRNA (short hairpin RNA) systems. Various vectors are used for efficient delivery of shRNA. Lentiviral vectors offer an efficient delivery system for stable and long-term expression of the shRNA in mammalian cells. The widely used lentiviral pLKO.1 plasmid vector is very popular in RNAi studies. A large RNAi database, a TRC (the RNAi Consortium) library, was established based on the pLKO.1-TRC plasmid vector. This plasmid (also called pLKO.1-puro) has a puromycin-resistant gene for selection in mammalian cells along with designs for generating lentiviral particles as well for RNA silencing. While using the pLKO.1-puro TRC control shRNA plasmid for transfection in murine P19 embryonic stem (ES) cells, it was unexpectedly discovered that this plasmid vector induced robust endodermal differentiation. Since P19 ES cells are pluripotent and respond to external stimuli that have the potential to alter the phenotype and thus its stemness, other cell types used in RNA silencing studies do not display the obvious effect and therefore, may affect experiments in subtle ways that would go undetected. This study for the first time provides evidence that raises concern and warrants extreme caution while using the pLKO.1-puro control shRNA vector because of its unexpected non-specific effects on cellular integrity.
RNA沉默被用作研究感兴趣基因功能缺失效应的常用方法。在哺乳动物细胞中，RNA干扰（RNAi）或RNA沉默可通过瞬时转染小干扰RNA（siRNA）或通过稳定的短发夹RNA（shRNA）系统来实现。各种载体用于高效递送shRNA。慢病毒载体为shRNA在哺乳动物细胞中的稳定和长期表达提供了一种高效递送系统。广泛使用的慢病毒pLKO.1质粒载体在RNAi研究中非常受欢迎。基于pLKO.1-TRC质粒载体建立了一个大型RNAi数据库，即TRC（RNAi联盟）文库。该质粒（也称为pLKO.1-puro）具有嘌呤霉素抗性基因，用于在哺乳动物细胞中进行筛选，同时还设计用于产生慢病毒颗粒以及进行RNA沉默。在使用pLKO.1-puro TRC对照shRNA质粒转染小鼠P19胚胎干细胞时，意外发现该质粒载体诱导了强烈的内胚层分化。由于P19胚胎干细胞具有多能性，并且会对外界刺激做出反应，这些刺激有可能改变其表型，进而影响其干性，而RNA沉默研究中使用的其他细胞类型并未表现出明显的效应，因此可能会以难以察觉的微妙方式影响实验。这项研究首次提供了证据，表明在使用pLKO.1-puro对照shRNA载体时应引起关注并格外谨慎，因为它对细胞完整性具有意外的非特异性影响。
