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GENE SILENCING WITH PCR PRODUCTS

east two different promoters and transfect them into the cells being targeted for gene knockdown (Figures 3, 4, 6, and 7). The promoter that is more effective for the siRNA and cell type will provide greater levels of gene silencing.


SEC Validation
The Silencer Express Kits have been used to create SECs targeting to GAPDH, c-fos, cyclophilin, PKC-alpha, -actin, and Green Fluorescent Protein (GFP). In all cases, SECs were able to reduce target gene expression. Figures 14 and 6 show several examples. Furthermore, SECs targeting GAPDH were functional in HeLa, 293, Cos7, Du145, HepG2, NIH3T3, MCF7 and A549 cells, confirming that PCR products can be transfected and transcribed in different cell types. Because they are simple to prepare, SECs provide an ideal way to screen potential siRNA sequences for the best siRNA sequence-promoter combination for DNA-based RNAi experiments in diverse cell lines. For additional information and data, click here.

Figure 6. Effectiveness of RNA Pol III Promoters with Altered 3' Ends. siRNA Expression Cassettes featuring the human U6 (Hu-U6) and human H1 (Hu-H1) promoters and expressing Negative Control (scrambled), GAPDH-, and cyclophilin-specific hairpin siRNAs were transfected into 293 cells. 72 hours post-transfection, the cells were processed to recover total RNA. Approximately 500 ng of total RNA from each sample was assessed by Northern analysis. Radiolabeled RNA probes targeting 28S rRNA, GAPDH, and cyclophilin were used to detect target genes and normalize levels of input RNA. Films were exposed to t
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