Figure 2.

LV-siGFP virus transduction. (a) Schematic drawing of lentiviral vector containing an siGFP cassette capable of generating siGFP-RNA. Shown also is the construct capable of generating two copies of siRNA after reverse transcription and integration. (b) Fifty thousand 293T cells were transduced with increasing amounts of LV-siGFP (15–375 ng of p24 per well). Fluorescence-activated cell-sorter analysis was performed to detect fluorescence. (c) Complete extinction of GFP expression by transduction of 80 ng (p24) of LV-siGFP virus in 5 × 10⁴ 293T-GFP-positive cells (×30). (d) 293T-GFP cells were transduced with increasing amounts of LV-siGFP (15–375 ng of p24 per well). Seventy-two hours later, whole-cell extracts were prepared, separated on 10% SDS/PAGE, and immunoblotted with polyclonal antibodies to GFP. As a control, 293T-GFP cells were also transduced with increasing amounts of LV-siHp53 and probed with GFP antibodies. β-Actin was used as a loading control.