This chapter describes the use of hTERT for the purpose of immortalizing primary human cells using the primary human fibroblast as an example. Tap dish to help dislodge cells, add 5 ml of 293T medium, and pipette up and down to break cell clusters. Viruses can be collected in three consecutive harvests over the next 24-72 h post-transfection. To collect viruses, replace the 293T medium with 4-5 ml of the target cell culture medium, in which the viruses will now be allowed to accumulate. Maintain the vector- and hTERT-transduced cells in continuous log-phase growth as cells are counted once a week. The presence of an active telomerase should yield a ladder of products with 6-bp increments, starting at 50 nucleotides. The lack of ITAS amplification would suggest that inhibitors of Taq DNA polymerase may have been present in some of the samples. If the assay has met these conditions, analysis of the experimental samples can now proceed.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)