Identification of Genes Regulating Gene Targeting by a High-Throughput Screening Approach
Figure 3
Cell-based assay for secondary screening. (a) Reporter system. The transgene used to assess I-SceI-induced gene targeting is integrated into the cGPS HEK-293 locus by expression of the I-CreI meganuclease. This transgene contains 1860 bp of the EF1α human promoter sequence, followed by (i) a puromycin cassette (used for selection), (ii) an IRES sequence, (iii) a GFP gene inactivated by the insertion of an I-SceI site generating a stop codon, (iv) a neomycin resistance sequence (used for selection) under the control of a CMV promoter. Repair plasmid and I-SceI induction (RMGFP+I-SceI) and repair plasmid alone (RMGFP) used for the induction of gene targeting by I-SceI are shown in the following, with the length of homologous sequence indicated. GFP reporter gene expression is assessed by flow cytometry for the quantification of gene targeting efficiency. (b) A clone resistant to both puromycin and neomycin was cotransfected with RMGFP+I-SceI or RMGFP, with or without siRNA, or with the following siRNAs: AS, LIG4, RAD51, and GFP. GFP was detected by flow cytometry 96 hours after transfection. The results of four independent experiments are presented.
