Figure 3
From: Short-term cytotoxic effects and long-term instability of RNAi delivered using lentiviral vectors
Shorter shRNA length reduced, but did not eliminate, cytotoxicity. A represents flow cytometry analysis of IS-1 cells 4 and 10 days after transduction with U6PT, sh319, sh321, sh323 and sh325 vectors. GFP expression is detected, and the percentage of GFP expressing cells was determined using the M1 gating shown (percentage GFP positive cells is shown in each histogram). B shows a comparative analysis of PAI-2 and OAS1 mRNA, in the samples described in A, 4 days after transduction. Data were generated by QRT-PCR and error bars are as described in previous figures. In C, cell lysates from samples of transduced cells described in A and B were subjected to immunoblotting with anti-PAI-2 monoclonal antibodies. Ponceau S staining served as a gel loading control, as did comparison of a single non-specific band (NS) in the immunoblot. D shows QRT-PCR analysis, as in B, for IS-1 cell mRNAs after transduction with or without U6PT, sh319, sh319scr and sh321 vectors.