Using a Lentivirus-Based Inducible RNAi Vector to Silence a Gene.

RNA interference (RNAi) is a powerful approach for inhibiting gene expression and its wide applications have expanded our understanding of gene functions. Short hairpin RNAs (shRNAs) are artificially synthesized RNA molecules used to mediate RNAi. The expression of shRNA in cells can be achieved by using plasmids or viral/bacterial vectors. The use of viral vectors to carry and deliver shRNA shows many advantages, including the ability to overcome the difficulty of transfecting certain cell types, the capability to establish stable cell lines via antibiotics selection or fluorescence-activated cell sorting, and the option to control temporally shRNA expression using inducible promoters. In this chapter, we introduce a gene silencing method utilizing a lentivirus-based inducible shRNA system. Using the human topoisomerase (TOP) gene as an example, we describe a procedure to generate stable HepG2 cells showing inducible suppression of TOP1. In addition, a procedure for assessing the efficiency of gene silencing is described in detail.