Selection of Potent Non-Toxic Inhibitory Sequences from a Randomized HIV-1 Specific Lentiviral Short Hairpin RNA Library
Figure 5
Expression of the shRNAs from different polymerase III promoters.
a) 6 potent shRNA sequences were subcloned to allow for U6 promoter driven shRNA expression. U6 or H1 promoter expression vectors and pNL4.3LucR-E- were co-transfected into HEK 293 FT cells. Western blot analysis with a HIV-1 Integrase specific antibody 48 h p.t. indicated that the shRNAs can be efficiently expressed from both polymerase III promoters. Actin was used as loading control. b) c) Published shRNA-sequences against pol, nef, rev/env, gag and vpu/env were cloned to be expressed by the U6 promoter. These constructs as well as 14 library shRNAs and pNL4.3LucR-E- were co-transfected into HEK 293 FT cells and analysed 48 h post transfection. Western blot analysis b) with a HIV-1 Integrase specific antibody or c) luciferase assays demonstrated that the newly identified shRNAs are as potent as the published shRNAs. A scrambled shRNA (sh scr) and non-transfected cells were used as controls.