Wang Xiaoyin, McManus Michael
Diabetes Center, University of California, San Francisco, USA.
J Vis Exp. 2009 Oct 2(32):1499. doi: 10.3791/1499.
RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are silenced at the post-transcriptional state. Short hairpin RNAs, precursors to siRNA, can be expressed using lentivirus, allowing for RNAi in a variety of cell types. Lentiviruses, such as the Human Immunodeficiency Virus, are capable to infecting both dividing and non-dividing cells. We will describe a procedure which to package lentiviruses. Packaging refers to the preparation of competent virus from DNA vectors. Lentiviral vector production systems are based on a 'split' system, where the natural viral genome has been split into individual helper plasmid constructs. This splitting of the different viral elements into four separate vectors diminishes the risk of creating a replication-capable virus by adventitious recombination of the lentiviral genome. Here, a vector containing the shRNA of interest and three packaging vectors (p-VSVG, pRSV, pMDL) are transiently transfected into human 293 cells. After at least a 48-hour incubation period, the virus containing supernatant is harvested and concentrated. Finally, virus titer is determined by reporter (fluorescent) expression with a flow cytometer.
RNA干扰（RNAi）是活细胞中的一种基因沉默系统。在RNAi中，与小干扰RNA（siRNA）序列同源的基因在转录后状态被沉默。短发夹RNA（siRNA的前体）可以通过慢病毒表达，从而在多种细胞类型中实现RNA干扰。慢病毒，如人类免疫缺陷病毒，能够感染分裂细胞和非分裂细胞。我们将描述一种包装慢病毒的方法。包装是指从DNA载体制备有活性的病毒。慢病毒载体生产系统基于一种“拆分”系统，其中天然病毒基因组已被拆分为单独的辅助质粒构建体。将不同的病毒元件拆分为四个单独的载体可降低通过慢病毒基因组的偶然重组产生具有复制能力的病毒的风险。在此，将含有感兴趣的短发夹RNA的载体和三个包装载体（p-VSVG、pRSV、pMDL）瞬时转染到人293细胞中。经过至少48小时的孵育期后，收集并浓缩含有病毒的上清液。最后，通过流式细胞仪检测报告基因（荧光）表达来确定病毒滴度。
