FIG 3.

Southern blot genotyping of targeted hESC clones and clonal variability of telomere length. (A) Telomere restriction fragment assay of the targeted hESCs over a time course after targeting. After digestion with MboI and AluI, 2 μg of genomic DNA was loaded into each lane and hybridized with a TTAGGG radioactive probe. Aberrantly targeted clones are marked with an asterisk. (B) Schematic overview of genotyping for N-terminal tagging at the endogenous TERT locus. Genomic DNA from single-cell-derived hESC clones was isolated and digested with either BamHI or HindIII. For genotyping, 5′-internal, 5′-external, and 3′-external probes were used. (C) Southern blot analysis for clonal Flag-TERT hESCs using three different probes. Clones 1, 5, 7, 11, 12, 13, and 15 were aberrantly targeted 5′ upstream of the right homology arm, as indicated with an asterisk. (D) Quantification of median telomere length from correctly genome edited individual clones (wt, n = 14; Flag, n = 11). P < 0.0001 (two-tailed Student's t test).