Figure 3.

The RNA construct, knockdown efficiency and mutants isolated during the siRNA-directed mutagenesis method. Cells harboring the HCV bicistronic full length replicon RNA shown in (A) were repeatedly electroporated with si18-36, si19-37 or si6367, and selected with G418 to pressure the selection of siRNA escape mutants. (B–D) Luciferase expression was assessed 3 days after siRNA electroporation for each iteration of the selection process to detect the evolution of knockdown resistance (left panel). Following seven rounds of knockdown and selection, the 5′ UTR sequence was amplified, cloned and sequenced. The sequence and incidence of mutations within the 5′ targeted site is shown for each siRNA used (right panel).