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Five Ways to Produce siRNAs

uch easier in otherwise hard to transfect cells.

Figure 3. Long Term Silencing of GFP with pSilencer 2.1-U6 hygro. HeLa cells expressing cycle 3 GFP were transfected with pSilencer 2.1-U6 hygro containing an insert encoding an siRNA targeting cycle 3 GFP or pSilencer 2.1-U6 hygro without an siRNA-encoding insert. Following transfection, the cells were selected with hygromycin. Three weeks following selection, the cells were analyzed for GFP expression by fluorescence microscopy. Green: GFP. Blue: DAPI stained nuclei. GFP levels were remarkably reduced (94%) in cells transfected with the GFP siRNA-encoding pSilencer 2.1-U6 hygro siRNA Expression Vector as compared to those transfected with an "empty" siRNA expression vector.


Method #5: siRNA Expression Cassettes
siRNA expression cassettes (SECs) are PCR-derived siRNA expression templates that can be introduced into cells directly -- without first being cloned into a vector. Initially described by Castanotto and colleagues (5), SECs include an RNA pol III promoter, a sequence encoding an siRNA hairpin, and an RNA pol III termination site. In contrast to siRNA expression vectors, which require cloning and sequencing prior to use and thus take 12 weeks to prepare, SECs can be generated by PCR in less than a day. SECs thus provide an excellent screening tool to find the most effective siRNA sequence, or to identify the most effective combination of promoter and siRNA in a given cell line. In fact, SECs provide the perfect complement to siRNA expression vectors. By incorporating restriction sites at their ends, SECs found to effectively elicit
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