hTERT immortalization - need help - (Dec/04/2008 )
I have a heterozygous knockout mouse and want to isolate primary endothelial cells for study. That's doable. But, once I get them, I want to create an immortalized cell line. That's what I don't know how to do. I've read that hTERT can be used but I know nothing about how to do this. Is there a company that sells any sort of kit to do this? Where do I start?
Get hTERT on an expression plasmid with selectable marker (e.g. G418) and transfect it into the cells, then select for transformants with the selective marker.
Overexpression of hTert alone may not guarantee immortalization. If you want to try, you can get hTert vector from Addgene.org for $75.
It is cell specific that whether hTERT alone could immortalize. For eg in humans, fibroblasts (lung, foreskin) can be immortalized by hTERT alone where as epithelial cells (except corneal) need abrogation of tumor suppressors. Check in literature for the cells which you want to immortalize. If hTERT alone is enough you can you retroviral vectors containing hTERT (Bob Weinberg or Sheila Stewart's lab plasmids in addgene), stably express the gene and immortalize. For abrogating pRb and p53 you can use SV40LTg.
also, www.atcc.org offers a hTERT immortalization kit
You are all fantastic - this is exactly the information and direction I needed to get going - Thanks!!