We have cloned our gene in mammalian expression vector pEGFN1 vector by introducing a stop codon between the gene of interest and EGFP sequence so that no GFP is expressed. While performing transient transfection in HeLa and A-549, we used pEGFPN1 vector as control (Where GFP is expressed). It has been observed that the vector is also showing toxicity but slightly lesser than the gene of interest containing pEGFPN1. This we got to know by doing MTT after 48 hours of incubation. Does anyone have any idea, how can I solve this problem? Or what is going wrong that can be corrected.
which chemical is using for your transfection? we are using Lipofectamine, due to this cell toxcity happening. i dont think a vector can make toxicity. it is due to tranfection reagent. So it can overcome by transfecting around 80% confluent cells. your gene of interest may fold some other way and it will lead to cell stress.
I agree that the toxicity you are seeing is most likely due to the transfection reagent, rather than the GFP (if I understand your question).
Depending on the reagent you are using, you can usually test different ratios of DNA:reagent, and you can certainly wash the reagent out after a period of time (say, 6/8 hours or so) if toxicity is high.
In our hands, transient transfection of plasmids always causes quite strong toxicity. If we want to overexpress a gene to perform functional characterization, we prefer to do either stable transfection using a selectable vector, or stable transduction with lenti- or retroviral particles. At least the cells will feel quite healthy then at the time you do your assay.
Good luck,
Cindy
I also meant to add that you can try changing the reagent. We have cell lines that transfect beautifully with Effectine, but also have other cell lines that will die quite spectacularly with the same reagent.
In my experience FuGene6 is quite good for avoiding toxicity, but as I say it's cell-line specific.
. @ Binshad . Thank you Binshad.. we are using lipofectamine 3000, as you said lipofectamine in itself have toxicity when used as control without plasmids. But the vector control and my gene of interest in vector have toxicity higher than the lipo control.
@Michael Porter.. Thank you Michael. I think washing is a good idea. Usually I just change the media without any washing.
@Cindy... thank you Cindy. we have tried establishing stable cell lines, but didn work out for this particular gene..
@Paritosh . We isolate plasmid using kit (Sigma) and for transfection we use Serum free media only
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