siRNAs Bypass the Antiviral Response
Interestingly, dsRNAs less than 30 nt in length do not activate the PKR kinase pathway. This observation, as well as knowledge that long dsRNAs are cleaved to form siRNAs in worms and flies and that siRNAs can induce RNAi in Drosophila embryo lysates, prompted researchers to test whether introduction of siRNAs could induce gene-specific silencing in mammalian cells (43). Indeed, siRNAs introduced by transient transfection were found to effectively induce RNAi in mammalian cultured cells in a sequence-specific manner. The effectiveness of siRNAs varies — the most potent siRNAs result in >90% reduction in target RNA and protein levels (44-46). The most effective siRNAs turn out to be 21 nt dsRNAs with 2 nt 3' overhangs. Sequence specificity of siRNA is very stringent, as single base pair mismatches between the siRNA and its target mRNA dramatically reduce silencing (44, 47). Unfortunately, not all siRNAs with these characteristics are effective. The reasons for this are unclear but may be a result of positional effects (46, 48, 49). For current recommendations on designing siRNAs, see "siRNA Design".

Although the history and mechanism of RNAi and PTGS are fascinating, many researchers are most excited about RNAi's potential use as a functional genomics tool. Already RNAi has been used to ascertain the function of many genes in Drosophila, C. elegans, and several species of plants. With the knowledge that RNAi can be induced in mammalian cells by the transfection of siRNAs, many more researchers are beginning to use RNAi as a tool in human, mouse and other mammalian cell culture systems.

In early experiments with mammalian cells, the siRNAs were synthesized chemically (Ambion is one of several companies that offer custom siRNA synthesis). Recently, Ambion introduced a kit (the Silencer™ siRNA Construction Kit) to produce siRNAs by in vitro transcription, which is a less expensive alternative to chemical synthesis, particularly when multiple different siRNAs need to be synthesized. Once made, the siRNAs are introduced into cells via transient transfection. Due to differences in efficacy, most researchers will synthesize 3–4 siRNAs to a target gene and perform pilot experiments to determine the most effective one. Transient silencing of more than 90% has been observed with this type of approach (44-46, 48, 49).

So far, injection and transfection of dsRNA into cells and organisms have been the main method of delivery of siRNA. And while the silencing effect lasts for several days and does appear to be transferred to daughter cells, it does eventually diminish. Recently, however, a number of groups have developed expression vectors to continually express siRNAs in transiently and stably transfected mammalian cells (50-56). Some of these vectors have been engineered to express small hairpin RNAs (shRNAs), which get processed in vivo into siRNAs-like molecules capable of carrying out gene-specific silencing (50, 53, 54, 56). The vectors contain the shRNA sequence between a polymerase III (pol III) promoter and a 4-5 thymidine transcription termination site. The transcript is terminated at position 2 of the termination site (pol III transcripts naturally lack poly(A) tails) and then folds into a stem-loop structure with 3' UU-overhangs. The ends of the shRNAs are processed in vivo, converting the shRNAs into ~21 nt siRNA-like molecules, which in turn initiate RNAi (50). This latter finding correlates with recent experiments in C. elegans, Drosophila, plants and Trypanosomes, where RNAi has been induced by an RNA molecule that folds into a stem-loop structure (reviewed in 3).

Another siRNA expression vector developed by a different research group encodes the sense and antisense siRNA strands under control of separate pol III promoters (52). The siRNA strands from this vector, like the shRNAs of the other vectors, have 5 thymidine termination signals. Silencing efficacy by both types of expression vectors was comparable to that induced by transiently transfecting siRNA.

The recent studies on RNAi have taken the research world by storm. The ability to quickly and easily create loss-of-function phenotypes has researchers rushing to learn as much as they can about RNAi and the characteristics of effective siRNAs. In the future, RNAi may even hold promise for development of gene-specific therapeutics. Much has been learned about this powerful technique, but additional information becomes available on an almost daily basis (see The RNA Interference Resource to learn about the very latest RNAi research and tools). It is not an understatement to say that the field of functional genomics is being revolutionized by RNAi.

上一页  [1] [2] [3] [4] [5] [6] 下一页