( l. 2001). The amount and location of ...)More siRNA Vectors for RNA Interference

l. 2001). The amount and location of siRNAs expressed from these two promoters is expected to vary from cell type to cell type. In studies comparing the expression of small RNAs under the control of the U6 promoter and several other RNA Pol III promoters, it was found that RNA synthesis can vary by orders of magnitude between the promoters depending on the cell line being used (Ilves et al. 1996). Furthermore, localization studies have shown that different RNA Pol III promoters generate small RNAs that localize to different sub-regions of cells (Ilves et al. 1996). The expression and localization of siRNAs will undoubtedly affect the success of RNAi studies in mammalian cells, making it important to test siRNAs under the control of both promoters.

In July 2002, Ambion introduced pSilencer 1.0-U6, which contains the mouse U6 promoter (Figure 1A).

Figure 1. pSilencer siRNA Expression Vectors; Maps and siRNA Design. For each target gene two complementary 55-60 mer oligonucleotides must be prepared. The oligonucleotides should encode the 19 mer hairpin sequences specific to the mRNA target, a loop sequence separating the two complementary domains, and a polythymidine tract to terminate transcription by RNA Pol III. The insert design shown above is specific for the pSilencer 2.0-U6 and 3.0-H1 Expression Vectors and contains the overhanging 5' ends to facilitate efficient and directional cloning into these plasmids. The insert for pSilencer 1.0-U6 would contain the appropriate end sequences for cloning into the Apa I and EcoR I sites. Earl
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