I have a non-human mammalian cell line (from a deer) that grows fairly well but is not immortalized. I want to do a CRISPR HDR integration and isolate clones to store for future experiments, which will require the cells to be grown for quite a few generations. I'm afraid the cells will become senescent before they replicate enough times to be useful.
I looked in AddGene for telomerase expression vectors but I seem to be finding only retroviral vectors, e.g. .addgene.org/1773/. We're not familiar with working with those. We were hoping to find a non-retroviral plasmid that we could use to integrate randomly into the genome and do selection with G418 or Hygromycin, as we've done with vectors from the pcDNA series of plasmids.
Is there some reason that non-retroviral plasmids can't be used for the purpose of immortalzing a cell line with telomerase?
Lentivirus and etrovirus are very effective in mammalian cell immortalization compared to plasmid. Plasmid exists in the cells for transient expression, immortalization requires stablely expression rather than transient, secondly, for primary cells plasmid transaction efficiency is low compared to viral vector. You can get ready to use lentivirus for hTERT. By the way, telomerase itself may or enough for some cells, you may need to add second gene like the large T antigen, also called SV40 gene. I used plasmids and failed many years ago. The virus is far more efficient for immortalization.
We're able to transfect these cells at a rate of >80% using a Lonza Nucleofector, so transfection efficiency doesn't seem like it would be a limiting factor. My intention was not to look for transient transfection, but to select stable transfectants with G418. Did you try drug selection?
The point about T antigen is interesting. I may want to plan to add both proteins, one way or another.
Thanks,
Mike
You might have to clone telomerase into the vector yourself, but since your cells sound highly transfectable you could probably introduce it via a PiggyBac. It probably would probably just involve taking an existing expression cassette vector where the expression cassette is flanked by PiggyBac repeats, cloning the telomerase into it that vector, then co-transfecting the expression cassette vector with a separate plasmid that has the PiggyBac transposase on it. That would avoid viral vectors and might be more efficient than random integration.
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