|
|
RNA interference (RNAi), a form of post-transcriptional gene silencing induced by introduction of double-stranded RNA (dsRNA), has become a powerful experimental tool for studying gene function. The RNAi phenomenon was first discovered in Caenorhabditis elegans and is characterized by sequence-specific gene silencing elicited by introduction of dsRNA (Fire et al. 1998; Elbashir et al. 2001) complementary to a target mRNA. In the endogenous RNAi pathway, long dsRNA is cleaved by the RNase III type endonuclease, Dicer, to produce 21–23 base pair (bp) short interfering RNAs. The siRNAs are in turn unwound and incorporated into a multiprotein complex known as the RNA-induced silencing complex (RISC), generating a sequence-specific nuclease that guides the cleavage of specific complementary mRNAs. In mammalian cells, direct introduction of siRNAs is used to experimentally initiate RNAi, because introduction of long dsRNA induces a potent antiviral response in addition to RNAi. | | Because of its ease of use, RNAi has been rapidly adopted for functional genomics, pathway analysis, and drug target validation experiments, and is now being used in high-throughput experiments with large numbers of siRNAs, or siRNA libraries. A key to all successful siRNA experiments is efficient delivery of the siRNA into cells and subsequent uptake of the siRNA by the RISC. RNAi can be successfully elicited in mammalian cells using exogenously derived siRNA only when the correct method and matrix of delivery conditions are employed for the cell type being used. |
|