Who killed my cells??? - Toxicity during transfection (Aug/01/2005 )

Hi,
I am having some problems in my transfection experiments, maybe someone has experienced the same and can give some hint...?

I have tried to transfect HepG2 cells with the empty pcDNA vector and also with the pcDNA containing my GOI. I obtained clones of cells after two weeks from start of selection. Cells containing the empty vector are
growing fine , but cells transfected with the GOI started to die after the two weeks .
All the conditions were the same for both transfections and posterior
treatment with geneticin was also identical .

Should one conclude that the overexpression of my POI was the cause of cell death?
Is it possible to avoid this by reducing the amount of DNA used(can the level of over expression be regulated in this way)??? Or is transient transfection the only solution?
I have also considered to clone my gene in a different vector for inducible expression, but that would supose starting again from 0.

Thanks in advance!

coco

If your empty vector cells survived the transfection and selection but your cells "containing" plasmid with GOI die ... looks as if your GOI is bad for your cells .

Inducible system may work ... allow you to get stable cells (if you use a non-leaky system). Transient may be easiest solution. Try transient first then look into inducible vectors.

If your empty vector cells survived the transfection and selection but your cells "containing" plasmid with GOI die ... looks as if your GOI is bad for your cells  .

Inducible system may work ... allow you to get stable cells (if you use a non-leaky system). Transient may be easiest solution. Try transient first then look into inducible vectors.

Hi,

thanks a lot for your answers.
I am now a little bit confused because I had the idea that selection with G418 takes a minimum of 2 weeks and máximum of 4, being slowlier on slowly growing cells. Am I wrong?

coco