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GENE SILENCING WITH PCR PRODUCTS

e U6 (Mo-U6), human U6 (Hu-U6), and human H1 (Hu-H1) promoters and encoding a -actin-specific hairpin siRNA were transfected into HeLa cells. 72 hours post-transfection, the cells were assessed using nuclear staining with DAPI and immunofluorescence using a -actin antibody. Non-transfected cells (NT) as well as cells transfected with an SEC expressing a negative control siRNA (scramble) provide wild-type levels of -actin. The relative level of reduction in gene expression was quantified and is provided in the bar graph.


Advantages of siRNA Expression Cassettes
In contrast to siRNA expression vectors, which require cloning and sequencing prior to use and can take 1-2 weeks to prepare, SECs can be prepared in less than a day. SECs thus provide an excellent screening tool to find the most effective siRNA sequence, or to identify the best combination of promoter and siRNA sequence in the experimental system being used. In fact, SECs provide the perfect complement to siRNA expression vectors. By incorporating restriction sites at their ends, SECs found to effectively elicit gene silencing can be readily cloned into a plasmid to create an siRNA expression vector. The siRNA expression vector can then be used for stable expression and long term studies.


Silencer Express Kits
Ambion's Silencer Express Kits provide the reagents necessary to prepare SECs with the Human H1, Human U6, or Mouse U6 promoters. Each kit includes a DNA fragment containing one of the
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