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GENE SILENCING WITH PCR PRODUCTS

siRNA Expression Cassettes


siRNA expression vectors offer enormous opportunities to study gene silencing. Unfortunately, the preparation of siRNA vectors is labor and time intensive. Ambion's new Silencer Express siRNA Expression Cassette Kits provide a rapid PCR-based method for preparing siRNA expression cassettes (SECs) that can be introduced into cells to induce gene silencing without prior cloning or sequencing. Because they are easy to prepare, SECs are ideal for screening target sites. Once effective SECs are identified, they can be subcloned into an appropriate vector for
subsequent gene silencing studies.


siRNA Expression Cassettes: How They Work
The use of PCR products to express siRNAs in mammalian cells was recently described by Castanotto and colleagues (1). These PCR products, referred to as siRNA Expression Cassettes (SECs), contain an RNA Polymerase III (Pol III) promoter adjacent to a hairpin siRNA template and an RNA Pol III transcription termination site. When transfected into mammalian cells, the SECs are transcribed by endogenous RNA Pol III to generate a small hairpin siRNA with several U's at the 3' end. The hairpin is thought to be processed by the dsRNA-specific Dicer enzyme (2) to generate functional siRNAs, which can enter the RNAi pathway and reduce the expression of a target gene. Figures 14 show examples of gene silencing experiments performed using the SEC approach.

Figure 1. siRNA Expression Cassette-Mediated Reduction

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