Identification of Genes Regulating Gene Targeting by a High-Throughput Screening Approach

Figure 4

Hits selected after secondary screening. Cells were cotransfected with 200 ng of RMGFP+I-SceI and a panel of different siRNAs, as indicated, at a final concentration of 33 nM. GFP fluorescence was detected by flow cytometry 96 h after transfection. The results are expressed as the stimulation factor, corresponding to the ratio of the percentage of GFP-positive cells in the presence of the siRNA hit to that following transfection with the control siRNA (AS). Four independent experiments were performed and Student’s 𝑡-tests were used to assess the significance of differences in stimulation factor (𝑃 value <  .05). Two siRNA controls were used to validate siRNA transfection: RAD51 and GFP siRNAs. The RAD51 and GFP siRNAs decreased the percentage of GFP-positive cells by factors of 6 and 4 with respect to the AS control siRNA, demonstrating that the siRNAs were active.