RNA Interference (siRNA, shRNA)

The discovery that gene expression can be regulated using small RNAs that are complementary to messenger RNAs - a process known as RNA interference (RNAi) - has markedly advanced the understanding of eukaryotic gene regulation and function. Notably, just one decade since its discovery, RNAi is already being tested in clinical trials and is set to revolutionize the treatment of disease. In recent years, much progress has been in improving the efficiency and widening the possibilities of RNAi. Lentiviral-mediated delivery of RNAi represents a major advance in the field. Despite the transfection of chemically synthesized small RNAs, lentiviral-mediated gene silencing can be applied not only to regular cell lines, but also to primary and nondividing cells. Furthermore, the transduced vectors can contain markers for selection, allowing the generation of stable cell lines, and also of cassettes to obtain a regulated shRNA expression. The protocol in this chapter describes the steps leading to the production of lentiviral vectors, and to infection of the desired recipient cells.