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Fig. 4 | BMC Biotechnology

Fig. 4

From: Targeted integration in human cells through single crossover mediated by ZFN or CRISPR/Cas9

Fig. 4

Southern blot analysis of integration events in single cell clones of ZFN-driven knock-in of only the EGFP donor plasmid. a The schematic diagram of detecting a 4-kb segment of 5′ junction DNA using Bam HI digestion and a 8.6-kb segment of 3′ junction DNA by using Hpa I digestion in single copy integration of donor plasmid. The binding positions of the probes (red line for 5′ junction analysis, and orange line for 3′ junction analysis) was indicated. b The schematic diagram of detecting a 4-kb segment of 5′ junction DNA and 6.4-kb donor plasmid fragment by using Bam HI digestion in multi-copy integration of donor plasmid. Red line indicates the binding site of probe. c The schematic diagram of detecting a 8.6-kb segment of 3′ junction DNA and 6.4-kb donor plasmid fragment by using Hpa I digestion in multi-copy integration of donor plasmid. Orange line indicates the binding site of probe. d Southern blot analysis of the 5′ junction of six single cell clones of ZFN-driven knock-in of only the EGFP donor plasmid. NC represents negative control, where genomic from untransfected cells was used for analysis. P represents positive control, where amplified PCR product using primers for preparing 5′ probe was used for hybridization. e Southern blot analysis of the 3′ junction of six single cell clones of ZFN-driven knock-in of only the EGFP donor plasmid. P represents positive control, where amplified PCR product using primers for preparing 3′ probe was used for hybridization

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