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GENE SILENCING WITH PCR PRODUCTS

three promoters, PCR primers specific to the 5' and 3' termini of the SECs, purification reagents to prepare SECs for transfection, and siRNA template oligonucleotides that can be used to generate positive and negative control SECs for siRNA studies. Both primers have modified ends to enhance SEC stability in mammalian cells (Figure 5). To facilitate cloning of the PCR products after testing, the 5' primer encodes an EcoR I restriction site and the 3' primer contains a Hind III restriction site. Ready-to-use pSEC receptor vectors with selectable markers will be available from Ambion soon. See the RNA Interference Resource at www.ambion.com/RNAi for more details.

Figure 5. Modifications in the Primers Provided with the Silencer Express Kit Produce siRNA Expression Cassettes (SECs) with Greater Stability in Cells. SECs designed to target GAPDH were prepared using the Silencer Express procedure from primers either with or without modifications to enhance stability. The SECs were transfected into 293 cells and RNA from the cells was analyzed after the indicated number of days. RNA was isolated with the RNAqueous Kit and then GAPDH mRNA levels were assessed by Northern blot using the NorthernMax-Gly Kit. The data are expressed as a percentage of GAPDH mRNA found in cells transfected with an SEC expressing a negative control (NC) siRNA. The modified SECs demonstrated a greater level of gene knockdown after 6 and 9 days. PCR primers contained in the Silencer Express Kits include the modifications used in this experiment.


Preparing SECs with the Silencer Expres
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