Identification of Genes Regulating Gene Targeting by a High-Throughput Screening Approach
Figure 1
A cell-based assay for identifying factors stimulating I-SceI-induced HGT. (a) A reporter system for I-SceI-induced gene targeting. The firefly luciferase gene (Luc2) is inactive due to replacement of the first 22 base pairs (bp) by a 24 bp I-SceI site (vertical black box). The plasmids constructed for I-SceI-induced gene targeting are described as follows: RMLuc+I-SceI has (i) the first 22 bp of the luciferase gene surrounded by 1 kb of homologous sequence (hatched boxes); (ii) an I-SceI induction cassette under the control of a CMV promotor. The RMLuc plasmid does not contain the I-SceI expression cassette. (b) Validation of the cell-based assay. The E2 cell line carrying a single integrated copy of the reporter system described in (a) was transfected with empty vector (pUC), RMLuc, or RMLuc+I-SceI (left panel). Luciferase activity was analyzed 72 hours after transfection. The E2 cell line was also cotransfected with either pUC or with RMLuc+I-SceI plus siRNAs known to modulate gene targeting (right panel): RAD51 siRNA and LIG4 siRNA. The results obtained were compared with those for cotransfection with the control All Star (AS) siRNA. Luciferase activity was assessed 72 hours after transfection. (c) Western blot analysis of Rad51 protein levels at various times after transfection, for the E2 clone with RMLuc+I-SceI and RAD51 siRNA. Specific and nonspecific bands were detected at 30 and 50 kDa, respectively.
