miR-Sens vector construction and validation. (A) Structure of the miR-Sens vector and sequence of the multiple cloning site (MCS). The MSCV packaging sequence between the 5′ LTR and the RL is not shown on this graph. The vectors are not to scale. (B) Flowchart for the validation of the miR-Sens vector. Three independent 293/T transfections with the three vectors (Vec_A, Vec_B, and miR-Sens) were done on three different days. Supernatants were collected on the third day post-transfection, and flash frozen. On the same day, the 293/T cells were harvested and equal amounts of cells were flash-frozen in liquid nitrogen. (C) Results of the 293/T cell transfection. The 293/T transfected cell pellets were tested in triplicate on the same day. (D) Results of the BJ cell transduction. BJ cells were transduced in triplicate on the same day with the three independent viral supernatants collected from the three 293/T cell supernatants. BJ cells were assayed 3 d after transduction as described in the Materials and Methods. Results correspond to the Mean ± SEM. (LTR) Long Terminal Repeat; (RL) Renilla luciferase; (PAS) synthetic PolyAdenylation Sequence; (T1) Expected Renilla luciferase transcript; (T2) Expected Firefly luciferase transcript; (TK) Thymidine Kinase promoter; (FL) Firefly luciferase; (SV40 PAS) Simian Virus 40 late PolyAdenylation Signal.
