triple stable transfection - (Oct/23/2007 )

Hi,
I'm trying to stable transfect in 3 plasmids into 293 cells at the same time. One of the plasmids is selected with hygromycin. However, 2 of the plasmids, pEGFP and pDsRed, both use the same antibiotic selection (G418). Does anyone know if this will be a problem? (Also, is it essential to linearize my plasmid before transfection?

Thanks,
joe

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if both the fluorescent vectors have the same antibiotic marker, then you might select cells which might have just one of the fluorescent plasmids and not necessarily both.

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Hi,

I agree with scolix.
The whole procedure will make your cells suffer from the selection and overexpression of selection markers and your 'target proteins'.
I guess you would preferably gain clones that express one or the other of the fluorescent proteins.

I'm no expert at all, but I would imagine that it is 'easier' to start with a double transfection, let the cells recover and stable transfect these cells again with a third plasmid.

Good luck,
Cheers

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As Scolix and Bomber said, cells resistant to G418 can be either doubly transfected or contain only one of your targets. Even if you do this transfection sequentially, your cell will already be resistance to G418.
as you want to generate stable lines i suggest to use flow cytometry, select you positive clones by drug selection first (so you want cells that are resistant to both antibiotics), let the cells recover and expand and sort them according to fluorescent. Those cells resistant to both antibiotics and positive for both green and red fluorescent will have been successfully transfected with the 3 plasmids.

hopes this makes sense.

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is it at all possible to move all your gene elements into 1 or 2 plasmids?

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So I tried transfecting all three plasmids. (pEGFP, pDsRed and protein expressing plasmid). The cells seems good for about 2 weeks, meaning there were cells that fluoresce both Red and green, and survived both G418 and hygromycin selection. However, after a couple more days alot of the cells seemed to die and very few cells expressed any fluorescence. If there was any it would be red but no green (meaning no EGFP expression). So it seems the best option would be to only transfect in two plasmids and leave one of the fluorescence proteins out. Also, I'm using 500ug/mL G418 and 200ug/mL hygromycin (based on a kill curve I ran). Are these the concentrations people use, I worried I may be using too much antibiotics. Also, Is it nessary to linearized my plasmid?

Thanks,
Joe

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for stable transfection, yes you do need to linearise your plasmid. Only a linear plasmid has the free ends neccessary for strand invasion and thus integration into the genome, unless of course you are using transposable elements and transposase to get your construct into the genome.

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First, lineariued plamid are not necessary but give better results. I did lots of stable transfections with just the uncut vector and it works fine.

Regarding your problem with 2 plasmid having the same selection marker:

To my experience this cannot work. Even if you would initially see green and red and the pasmid would be integarted into the genom, the cells would need only one copy of the resistance marker and therfore by the time lose one copy again resulting in a fade or loss of either red or green fluorescence over time.

I have even seen that cells stay resistant to the marker but selectively lose the fluorescence if transfected with only one plasmid.

So you need to use 3 selection markers for 3 plasmids. There are so many others besides G418 and hygromycin: for example blasticidin and zeocin.

The other option would be to use an IRES vector where you can express two genes independently (not as fusion proteins) at the same time. But also here I have seen results from colleauges where one of the genes was downregulated after a while...

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