ABSTRACT
Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Posttranscriptional gene silencing by RNA interference (RNAi) appears a promising, novel approach for the targeted inhibition of gene expression in cell culture and in vivo.1-3 RNAi can be mediated by exogenous delivery of small interfering RNA (siRNA), short duplexes RNA about 21mers in size that usually house two nucleotide overhangs at each 3'-end terminus. Inside cells, these duplexes are recognized by the RNA-induced silencing complex (RISC) in the cytosol, whereby the antisense RNA strand is then guided by this complex to the complementary sequence in target mRNA. Provided the complementary sequence in the target mRNA is hybridization accessible, the RISC complex then, by mechanisms that are not fully understood, initiates its hydrolysis and thus prevents or “silences” gene expression. This approach has been investigated for potential use as a biological tool for understanding gene function, as a drug target validation strategy, and as a potential therapeutic agent for the treatment of genetic diseases.1-3
Although endogenous expression of double-stranded RNA or short-hairpin RNAs using viral vectors has been successfully used for gene silencing in cells and in animal models,3 in this chapter we will focus on exogenous delivery systems for effective siRNA uptake into cells. In particular, we provide generic protocols for optimizing the uptake of synthetic siRNA using any delivery system, though we used cationic lipid formulations as a working example.
