Functional Identification of Cancer-relevant Genes through Large-Scale RNA Interference Screens in Mammalian Cells

Abstract

The discovery of a cellular response against doublestranded RNA (Fire et al. 1998) has provided one of themost powerful tools to manipulate gene expression andthis has revolutionized loss-of-function genetics inCaenorhabditis elegans and Drosophila (Ashrafi et al.2003; Kamath et al. 2003; Lum et al. 2003). In mostmammalian cells, however, the introduction of long double-stranded RNA provokes an interferon response, leading to a general shutoff of protein synthesis (Stark et al.1998). This response can be bypassed by using chemically synthesized, 21-base-pair double-stranded short interfering RNA (siRNAs), which can cause strong, buttransient, inhibition of gene expression in nearly all mammalian cells (Elbashir et al. 2001). Indeed, small-scalegenetic screens with sets of in vitro synthesized siRNAshave recently been performed in mammalian cells toidentify modulators of apoptosis (Aza-Blanc et al. 2003).However, the use of these siRNAs is limited by the transient inhibition of gene expression and their high cost. Tocircumvent these limitations of siRNAs, we and othershave developed expression vectors that direct the synthesis of short hairpin RNAs (shRNAs), which are processedin vivo to siRNA-like molecules that can suppress geneexpression over prolonged periods of time (Brummelkamp et al. 2002b; Miyagishi and Taira 2002; Paddison et al. 2002; Sui et al. 2002; Yu et al. 2002). We haverecently shown the feasibility of using shRNA vectors toidentify loss-of-function phenotypes in mammalian cellsby creating a set of vectors to suppress nearly all members of the family of de-ubiquitinating enzymes. Usingthis approach, we identified the cylindromatosis tumorsuppressor gene as a key regulator of the transcriptionfactor NF-κB (Brummelkamp et al. 2003). More recently,we constructed a large set of shRNA vectors that togethertarget some 8000 human genes for suppression (Berns etal. 2004). We discuss here several ways in which suchshRNA vector libraries can be used to identify novelcomponents of cancer-relevant pathways...