RNAi

The discovery that dsRNA could act as a potent and specific mediator of gene silencing has led to a revolution in the use of RNAi to determine the function of genes and to identify potential drug targets in mammalian cells (64). Based on an understanding of the RNAi mechanism and testing of multiple synthetic siRNAs as well as gene-expressed shRNAs to several target RNAs, design rules and associated algorithms have been developed for identifying potential RNAi-sensitive sites on target RNA (88, 89). In most instances, systematic analysis of three different siRNAs, or shRNAs, per target identified an effective gene-silencing agent that reduces target RNA levels by between 50 and 70%. A variety of methods can be used for the delivery of synthetic siRNAs, and several different vector systems exist for expression of shRNAs in mammalian cells (68, 90). This form of gene silencing has been applied to a range of different molecular and biological targets, most of which have been exhaustively reviewed (68). One potentially attractive feature of RNAi for gene functional studies is the capacity to generate an epi-allelic series of cell clones for a specific target gene using shRNA-expression vectors (91). In addition, the ability to express shRNAs that undergo processing to direct RNAi-mediated gene silencing of specific target RNAs has inspired the development and testing of longer dsRNAs in mammalian cells, with the aim of producing multiple siRNA products (92). Theoretically, these multidomain dsRNA hairpins could be used to produce multiple siRNAs to a single target or to generate single siRNAs to many different targets. The current limitations with RNAi are the potential for off-target effects and induction of the interferon response due to the promiscuous nature of RNA-mediated gene silencing for some target sites (see (93) for comment). This remains controversial and will require further experimental studies.

An exciting extension of posttranscriptional silencing by siRNAs is the potential for inducing sequence-specific transcriptional silencing using the RNAi machinery (94). RNAi-mediated DNA methylation and associated transcriptional silencing was first demonstrated in plants (95). More recently, two independent studies have shown that synthetic siRNAs or gene-expressed shRNAs directed against mammalian promoters, and localized to the cell nucleus, could induce DNA methylation at their associated target sites in the genome (96, 97). This approach to gene silencing in mammalian cells may provide an alternative route for inactivating specific gene expression at the level of the gene itself.

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