BALB/c 3T3 transfection - (Oct/07/2006 )

Hi all

I was just wondering if anyone has had success with stable transfection of BALB/c 3T3 fibroblasts?? I haven't been able to get a very high transfection efficiency (less than 20%) and my cells are losing expression of my EGFP fusion protein within 3 weeks (under G418 selection).

Also, I find that I have many live, non-fluorescing (non-transfected) cells after 3 weeks. I can't thinks of an explanation for this as I have titrated my stock of G418 twice to make sure I was using a sufficient amount to kill sensitive cells (I am using 1mg/ml)

Any ideas??

thanks

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Hi Laura,

The most likely explanation is that the EGFP fusion protein is disadvantageous to the growth of your culture. -Hence, you will have a selective pressure favouring cells that "close down" expression of your EGFP fusion, while maintaining the antibiotics selection.
-this is notoriously happening in 3T3 fibroblasts (especially when dealing with nuclear regulators....)
Regarding the low transfection efficiency: actually 20% is a very high efficiency in 3T3 cells (!)
-But I would forget about transfection and simply go for transduction (usually gives effieciencis of 80-95%). And for 3T3 cells the pBABE vector system appears especially usefull.

Hope this is helpfull! and wishing you Good luck!

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Thanks for your suggestions, Dr dandy

Just another question though
Is it possible to retain G418 resistance and shut down EGFP expression if they are both under the same promoter??

Thanks again

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