Figure 5

Reversibility of telomerase activity via the hTERT binary vector. Lysates from hTERT-OFF(a) cells shown in Figure 4 were assayed for telomerase activity by the telomere repeat amplification protocol (TRAP). One microgram total protein was used for each sample, with a 25-cycle amplification step (see Materials and Methods). Lane 1, no lysate added. Lanes 2–7, lysates from HA-5 cells containing the hTERT-OFF vector, followed by introduction of pUC (lane 2) or Cre recombinase (lanes 3–7), at the indicated population doublings. Lane 8, an unrelated population of HA-5 cells containing stably expressed hTERT. Lane 9, a population of untreated HA-5 cells split following introduction of hTERT-OFF, at population doubling 65. Lanes 10–12, untreated HA-5 cells, containing no additional vector sequences, at the indicated population doublings. The asterisk indicates a PCR product that serves as an internal positive control for the PCR reaction itself. At left, a bracket shows the amplified telomeric repeats indicative of the presence of telomerase activity.