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. Author manuscript; available in PMC: 2010 Oct 9.
Published in final edited form as: Biochem Biophys Res Commun. 2009 Jul 25;388(1):56–61. doi: 10.1016/j.bbrc.2009.07.112

Figure 2.

Figure 2

Efficiency and efficacy of ZFN-mediated gene correction in HEK293 cells using 4-finger CCR5-specific ZFNs fused to different FokI nuclease domain variants. Panel (A) brightfield; Panel (B) GFP positive cells seen post-transfection of HEK293 Flp-In cells with 4-finger CCR5-ZFNs and donor plasmids; Panel (C) FACS analyses (Q4 shows GFP+Cells). WT-FN, CCR5-ZFN constructs carrying the wild-type FokI nuclease domains. OHD depicts obligate heterodimers. OHD1-FN, CCR5-ZFN constructs carrying the FokI nuclease domain mutants that were generated at PBPL. OHD2-FN, CCR5-ZFN constructs carrying the FokI nuclease domain mutants reported by Szczepek et al., 2007 (20). OHD3-FN, CCR5-ZFN constructs carrying the FokI nuclease domain mutants reported by Miller et al., 2007 (19). (D) Frequency of gene correction in HEK293 Flp-In cells of a chromosomal mutant GFP reporter disabled by insertion of the CCR5 ZFN target sequence. Quantitative FACS analyses of the GFP positive cells at 3, 5 and 7 days post-transfection with designer ZFNs and donor plasmids. WT-FN, ZFN constructs carrying the wild-type FokI nuclease domains. (E) Analysis of the genotype of four different individual GFP positive clones. Five days post-transfection with ZFNs and the donor plasmids, GFP positive cells were sorted, serially diluted to get individual clones and grown. The genomic DNA was isolated from the GFP positive clones and the eGFP gene at the Flp-In locus was PCR amplified and digested with BstXI. The mutant eGFP gene has two BstXI sites, where the ZFN binding sites are inserted. Correction of the eGFP gene by homology-directed repair results in the loss of the BstXI sites. The PCR product size of the corrected eGFP gene is 930 bp as compared to 990 bp for the mutant gene. BstXI digestion of the mutant eGFP PCR product generates two bands: 450 bp and 540 bp, respectively. Lanes: Control, PCR product of the mutant eGFP gene from untransfected cells before (-) and after (+) digestion with BstXI; GFP+1-4, PCR products of 4 different individual clones obtained from GFP positive sorted cells before (-) and after (+) digestion with BstXI; M, 1 Kb ladder. All GFP positive cells are resistant to BstXI digestion, confirming ZFN-mediated eGFP gene correction in these cells.