stable transfection of two plasmids? - (Sep/19/2005 )

Now I want stable transfection of two plasmids. is it normal to transfect both once together or transfect oneplasmid, after get stable lines then transfect another one?
Never did two plasmids before, so no idea at all.
Thanks a lot
cathy

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I've never done it myself, but in my lab we use a few different cell lines that are stables with 2 different plasmids. One cell line is a tet-inducible using the tTA system and another just overexpresses two different oncogenes. I am pretty sure both were generated by two consecutive transfections, not transfecting both plasmids at the same time. Hope this helps a little. . .

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Try separate experiments, one in which you co-transfect both plasmids and two others in which you transfect both plasmids consecutively (after generating a stable one for your first plasmid you transfect the second one) and let us know your findings. If I'm not mistaken transiënt co-transfection in itself is quite efficiënt so it should be possible to get both plasmids in your cells at once (I'm not sure about stable co-transfection).

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Thank Vairus for your perfect explanation.

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idf you in hurry, you can try selection of two-gene stable transfectants but have to use different selection markers f.i. antibiotic resistance genes;


if you have more time, do sequential selection as parallel slection may to much stress for the cells although some may survive selection...

consider that the kind of transfected genes and their products may contribute to reduced survival fitness...

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