Short Hairpin Activated Gene Silencing in Mammalian Cells

RNA interference (RNAi) is now a popular method for silencing gene expression in a variety of systems. RNAi methods use double-stranded RNAs (dsRNAs) to target complementary RNAs for destruction. In mammalian systems, very short dsRNAs (22–25 bp) such as short interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) are used to avoid endogenous nonspecific antiviral responses that target longer dsRNAs. siRNAs elicit a transient silencing response, while shRNAs can be expressed continuously to establish stable gene silencing. shRNAs can be introduced into cells and animals using a variety of standard vectors as well as retroviral or lentiviral expression systems. This chapter describes the design, construction, validation, and use of shRNAs for silencing genes. We report our results from testing a variety of shRNA design features and shRNA expression vectors. We also provide methods that use shRNAs to permit different levels of gene expression. Additionally, we discuss some aspects important for constructing an information pipeline to support development of a large shRNA library.