Fig. 1. Generation of mTERT-overexpressing ES cells. (A) Expression of telomerase was determined by RT-PCR in vector-transfected and mTERT-expressing ES cells. 3 µg total RNA was reverse-transcribed into cDNA and two sets of primers were used to amplify the genes encoding mTERT and tubulin simultaneously. (B) Protein level of mTERT was determined by western-blot analysis using 100 µg nuclear extract from vector-transfected and mTERT-expressing ES cells. Topoisomerase-I levels were also determined, which represent loading in each lane. (C) Telomerase activity of vector-transfected and mTERT-expressing ES cells was determined by a TRAP assay. Decreasing amounts of protein (50 ng, 10 ng, 2 ng and 0.4 ng) were used in the assay.