FIG 2.

Endogenous tagging of TERT disrupts telomere maintenance and results in telomere length shorter than that seen with wild-type hESCs. (A) Relative expression levels of TERT and OCT4 mRNA in the endogenously epitope-tagged hESCs compared to isogenic wt cells measured by quantitative RT-PCR. Expression levels of TERT and OCT4 were normalized to GAPDH. n.s., P > 0.05; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001 (two-tailed Student's t test). (B) Telomeric repeat amplification protocol (TRAP) assay of whole-cell extracts from nonclonal wt, Flag, Flag-GS10, Flag-GS20, HA, and YbbR hESCs using decreasing amounts of protein (200, 40, and 8 ng). TRAP signals relative to those of wt hESCs were quantified and are shown at the bottom of the lanes (40 ng). IC, internal control. (C) Telomere restriction fragment assay of the targeted bulk population hESCs over a time course after targeting (days 37, 59, 80, and 100 after the second targeting). Restoration of telomerase after the second targeting resulted in substantial telomere elongation and an overall increase in telomere signal intensity. After digestion with MboI and AluI, 2 μg of genomic DNA was loaded in each lane and hybridized with a TTAGGG radioactive probe. (D) Quantification of median telomere length shown in panel C. (E) Telomere restriction fragment assay, as in panel C, of three independent editing experiments. Samples were collected at days 51 (left gel), 53 (middle), and 37 (right) after the second targeting.