A high throughput system to generate pHippy expression cassettes. a, A multiple primer PCR system was used to generate PCR products that express siRNA after transfection into cells. A 5' primer (U6 primer) was designed to human U6. A second primer (gene specific) was designed with sequences that are complementary to the 3' end of the human 6 promoter, the target siRNA sequence, and a sequence that is complementary to the 3' of the human H1 promoter. A third primer was designed that contains the human H1 promoter and is complementary to the gene specific primer. b, An agarose gel stained with ethidium bromide demonstrates the robustness of the PCR method for several different gene specific primers. c, A luciferase assay as described in the legend to Figure 3b to determine if the PCR products inhibit PGL3 luciferase activity.
Kaykas and Moon BMC Cell Biology 2004 5:16 doi:10.1186/1471-2121-5-16