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More siRNA Vectors for RNA Interference


Small Interfering RNAs (siRNAs) are short, double-stranded RNA molecules that can target complementary mRNAs for degradation via a cellular process termed RNA interference (RNAi) (Elbashir et al., 2001a). Recent publications have described the use of vectors expressing functional siRNAs to enable long-term knockdown studies (for a review, see Tuschl, 2002). Two different RNA Polymerase III promoters, the well-characterized U6 and H1 promoters, have been used in these vectors to drive the expression of siRNAs (Sui et al., 2002, Brummelkamp et al., 2002). Below we discuss the reasons why these two promoters were chosen and introduce two new siRNA expression vectors for RNAi experiments in mammalian cells.

The selection of RNA Polymerase III to drive siRNA expression derives from the facts that it expresses relatively large amounts of small RNAs in mammalian cells and that it terminates transcription upon incorporating a string of 3-6 uridines. The latter feature is especially important due to the apparent requirement that siRNAs have defined 3' termini that hybridize to an mRNA target (Elbashir et al. 2001b). Type III promoters have been selected for siRNA vector design because they are relatively simple and they lie completely upstream of the sequence being transcribed. This eliminates any need to include promoter sequence in the siRNA.

The sequences of the U6 and H1 promoters are very distinct, with the U6 promoter stretching across several hundred base pairs (Kunkel 1989) and the H1 promoter barely exceeding 100 (Myslinski et a
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