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GFP vector as a transfectino efficiency indicator?


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#1 neuronzy

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Posted 12 June 2011 - 09:11 PM

Hi!
I'm using FACS to test the DNA content (for cell cycle and apoptosis) of cells transfected with GFP-tagged vector expressing interest gene. I read in a paper that Us9-GFP vector and p53 expressing vector were co-transfected into cells and the GFP was used as a transfection efficiency indicator. I'm wondering that do the GFP-positive cells necessarily contain the vector of interest gene? Or is this the specificty of Us9-GFP? May I use pAcGFP1-C1 and p53 expressing vector to do this thing? I'm doing with a GFP-fused p53 vector, which confirms the coexistence of GFP and gene of interest, but I'm afraid that the fusion protein influences the basic function of p53.
Thanks!

#2 Rsm

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Posted 13 June 2011 - 10:43 PM

Yes, co-expression is very likely when you co-transfect. R-square is somewhere around 0.999, at least for lipofection. You can also use 2A peptides (or IRES) to improve this value and reduce the number of plasmids you use.
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#3 neuronzy

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Posted 14 June 2011 - 02:31 AM

Yes, co-expression is very likely when you co-transfect. R-square is somewhere around 0.999, at least for lipofection. You can also use 2A peptides (or IRES) to improve this value and reduce the number of plasmids you use.


Thank you!
I tried using IRES2-EGFP but due to the low translation efficiency by IRES, the fluorescence is very weak that it can not be analyzed by the flow cytometer in our institute.
Does different ratio of the plasmids (say 0.5ug:1ug or 0.75ug:0.75ug) affect the R-square? Besides, I perfrom transfection by using PolyJet reagent. Will the coordination change using different transfection reagents?

#4 Rsm

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Posted 14 June 2011 - 10:56 PM

My experience is with FuGene6 and equimolar ratio. You may want to test it yourself beforehand.
FACS is usually a very sensitive means of detection, I am a bit worried that you could not get any signal from your transfection. This may suggest that your efficiency is rather low (or you have a problem with your GFP).
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