Figure 1
From: AAV-mediated in vivo knockdown of luciferase using combinatorial RNAi and U1i
In vitro knockdown of luciferase by shRNA and U1i constructs. (a) Increasing amounts (1–250 ng) of shRNA constructs targeting firefly luciferase (shLuc1 and shLuc2) were co-transfected with 2.5 ng firefly luciferase and 0.5 ng renilla luciferase plasmid in 96-well plates containing approximately 2.5 × 104 HEK293T cells. The total amount of transfected DNA was kept constant by adding the pSuper cloning vector. At 2 days after transfection, cells were lysed and firefly and renilla luciferase signals were measured. Relative luciferase activity was calculated as the ratio of firefly and renilla luciferase and plotted relative to the 100% control transfected with 250 ng pSuper. shLuc1 significantly reduced luciferase by 85%, whereas shLuc2 was inactive. Control shRNA plasmids were shGFP and shApoB. (b) Co-transfection of 200 ng U1i constructs targeting firefly luciferase (L1-L8) with firefly luciferase and renilla luciferase plasmid. Cells transfected with 200 ng control plasmid U1C were set at 100%. L4 and L5 mildly reduced luciferase expression by 30 and 40%, respectively, compared with U1C. (c) Co-transfection of 100 ng L4 and 100 ng L5 significantly improved luciferase inhibition, compared with transfection with 200 ng L4 or L5 alone (P<0.05). Data are presented as mean of three technical replicates±s.d.
