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Immortalizing MEFs with SV40


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#1 Wek

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Posted 08 January 2016 - 11:16 PM

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

 



#2 bob1

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Posted 11 January 2016 - 12:11 PM

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.



#3 Wek

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Posted 12 January 2016 - 07:17 AM

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



#4 bob1

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Posted 12 January 2016 - 04:31 PM

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.



#5 saharmalu

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Posted 17 March 2016 - 03:49 PM

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






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