Immortalizing MEFs with SV40 - (Jan/09/2016 )

I would like to create a fibroblast cell line but I am unsure about some stuff. I want to keep things simple so I plan to obtain MEFs from mouse embryos and transduce them with SV40. Can I use pBABE-puro SV40 LT as my SV40 source? I dont want to buy an immortalization kit since they are crazy expensive.

My MEF isolation protocol uses MEM + supplements for culturing these cells. Could I use DMEM instead? I plan to package the virus with phoenix cells and they are grown in DMEM. I would like to grow both cell lines with the same medium but I dont know if the extra glucose would have a negative effect on the MEFs. 

Once immortalized I plan to transduce these MEFs again.

I would appreciate if someone with experience can offer some advice and answer these questions.

Thanks

IIRC you just need the LT antigen to cause immortalization, small t antigen probably helps as well, you could just express those from a plasmid and then select.

bob1 on Mon Jan 11 20:11:14 2016 said:

IIRC you just need the LT antigen to cause immortalization, small t antigen probably helps as well, you could just express those from a plasmid and then select.

So any plasmid that expresses the LT antigen will be sufficient to immortalize cells?

Thanks

Potentially, yes. You would want a method of maintaining the plasmid in the cells, so you need a selectable marker. Check out the paper linked on the Addgene page for your pBABE-puro construct... it has a lot of good info.

So any plasmid that expresses the LT antigen will be sufficient to immortalize cells?