Figure 1.

Construction and characterization of telomerase-rescued X-DC fibroblasts. (A) Schematic of retroviral infection. (B) Whole cell extracts from each X-DC cell line, expressing TERT alone, or both TERT and TER, were normalized according to protein concentration and assayed for telomerase activity using TRAP. Comparison of TRAP activities in X-DC-TERT cells without the co-expression of recombinant TER with WT cells expressing TERT showed that telomerase activity was reduced more than 2.5-fold. Telomerase activities of the TERT and TER expressing X-DC cells were all similar or higher than the levels observed in WT cells expressing only TERT. (C) Terminal restriction fragment analysis. X-DC cells, expressing TERT alone or both TERT and TER were grown in continuous culture and telomere length profiles were obtained using Southern blot analysis. In contrast to WT cells, X-DC cells expressing TERT alone exhibited a proliferation-dependent loss of telomere repeats. Stable telomere maintenance was restored in X-DC cells expressing both TERT and TER. (D) Growth characteristics of telomerase-rescued X-DC cells. All TERT/TER expressing X-DC cells were grown in continuous culture for many population doublings beyond what is shown. There were no observable changes in morphology or other growth characteristics.