Abstract
RNA interference was originally described as a powerful tool to inhibit gene expression in model organisms. Until recently, loss-of-function genetic screens in mammalian cells were hampered by a lack of suitable tools that can be used in a high-throughput format. Here we discuss the construction of short-hairpin RNA (shRNA) vector libraries, in particular those generated at the Netherlands Cancer Institute (NKI), and their application in mammalian cancer genetics. We describe their virtues and limitations, as well as different options for screening such libraries.