Figure 4.

Co-expression of RNAi and U1i from one vector backbone suppresses luciferase activity in vitro. Increasing amounts (25–250 ng) of shRNA/U1i plasmid expressing both shRNA and double U1i constructs were co-transfected with firefly luciferase and renilla luciferase plasmid and analyzed as described in Figure 1a. The total amount of transfected DNA was kept constant by adding the pro-AAV cloning vector. Cells transfected with 250 ng pro-AAV vector were set at 100%. The control vector AH expressed shApoB and double H1/H4. The U1i vector AL expressed shApoB and double L4/L5. The shRNA vector LH expressed shLuc1 and double H1/H4. The shRNA and U1i combination vector LL expressed shLuc1 and double L4/L5. AL, LH, and LL significantly reduced luciferase activity compared with the AH control vector (P<0.001) and LL was most effective and led to 85% luciferase knockdown. Data are presented as mean of three independent experiments±s.d., analyzed using factor correction.³⁶