Figure 3

A. Suppression of EGFP expression by a "YIU" shRNA construct in mouse NIH3T3 cells. This shRNA cassette targets EGFP nt 243–261, GCACGACTTCTTCAAGTCC, selected as an effective shRNA cassette by a screen described in the text. When cotransfected with pEGFP vector at a molar ratio of 3 to 1 (shRNA:pEGFP), GFP fluorescence was suppressed; DsRed2-C1 was used as an internal control. Images were taken 24 h after transfection. B. Quantitative analysis of suppression efficiency by flow cytometry. The shRNA expression vector and the GFP reporter vector pMIG, at a molar ratio of 3 to 1, were cotransfected into mouse NIH3T3 cells. A "classic" shRNA expression construct targeting luciferase was similarly cotransfected in control cells. Assays were performed in triplicate at 48 h after transfection. The gate M1 was established using untransfected cells. The suppression, as measured by the decrease in mean fluorescence of the gated population, was quantified by flow cytometry in triplicate for each sample. Note that this is likely to provide a minimal measure of suppression, as GFP expression of many transfected cells may no longer fall within the gate; in fact, the gated population is markedly decreased in cells cotransfected with the shRNA construct targeting EGFP.