Repair at Single Targeted DNA Double-Strand Breaks in Pluripotent and Differentiated Human Cells
Figure 1
A. A DSB formed by I-SceI within the SceGFP gene in p84-DR-GFP can either be repaired by nonhomologous end-joining, homologous recombination or single-strand annealing. HR using the iGFP template establishes a functional GFP. Precise NHEJ or HR using the SceGFP on the sister chromatid as a template can reestablish the I-SceI site. Arrows indicate primers for confirming targeting to p84. B. PCR demonstrates targeting at the 5′ and 3′ borders of p84-DRGFP. C. FISH demonstrates overlap of the p84 locus (green) and DR-GFP (orange) probes indicating targeting of DRGFP to the p84 locus in hESCs and HCT116 cells. Insets are magnified images of the indicated regions. D. Immunofluorescence microscopy for pluripotency markers in H9 hESCs, U20S and 293T cells.