Iain D.C. Fraser
Alliance for Cell Signaling, Molecular Biology Laboratory
California Institute of Technology, Pasadena, CA
Introduction
RNA interference (RNAi) has recently emerged as a powerful experimental tool in mammalian cell biology. Double-stranded RNA (dsRNA)-induced gene silencing was originally discovered as a mechanism for regulating expression of endogenous genes in C. elegans (1), although a similar phenomenon, termed cosuppression, had been demonstrated in plants (2). Studies of the RNAi mechanism demonstrated that the degradation of the target mRNA was mediated by short fragments of dsRNA (3, 4),which were shown to be derived from the longer dsRNA template through the action of the ribonuclease dicer (5). These short interfering RNAs (siRNAs), when exogenously introduced, can enter the RNAi pathway downstream of dicer and efficiently reduce expression of their target gene. The RNAi pathway was not initially considered a useful experimental tool in mammalian cells due to interferon responses to the dsRNA trigger(6). However, Tuschl and colleagues hypothesized that this approach could be applied to mammalian cells if introduction of the shorter siRNAs could avoid activation of the
interferon pathway (7). They confirmed their hypothesis in several mammalian cells lines(7), and RNAi has now been shown to be a valuable experimental tool in a variety of biological systems (8).
The direct transfection of chemically synthesized siRNA duplexes into mammalian cells, as originally demonstrated by the Tuschl lab, is currently the most
popular approach to RNAi. However, the applicability of this technique is dependent on the transfectability of the model cell system, and since the presence of the siRNA in the cell is transient, longer term experiments are more difficult. This issue has been addressed by the development of approaches that permit the expression of siRNAs from DNA-based plasmid vectors. Several groups have shown that siRNAs can be transcribed as stem-loop hairpin structures under the control of RNA polymerase III (pol III)promoters (9-13). Figure 1 shows a schematic of how such a short hairpin RNA (shRNA) can be transcribed from the promoter for the RNA component of RNase P, H1. Similar expression cassettes have also been developed using the promoter for the small nuclear RNase, U6.
