Hello all,
I'd like to know whether any of you have ever experienced lower transfection efficiencies of a bicistronic overexpression vector in mammalian cells carrying no insert 5' of the IRES (3' is GFP), compared to the vector containing a 4.5kb insert 5' of the IRES?
The Promotor is CMV, and the plasmids have been tested using 4 different transfection reagents in 2 different cell lines.
Transfection efficiencies were determined 36 - 48hrs after transfection by FACS detection of GFP.
For empty vector and vector+insert, efficiencies ranged from 10 to 40%, roughly, with the empty vector displaying lower effeciencies (10-15% less) in most cases. Even in HEK293H, transfection efficiencies were as low as 30 to 40% for both vectors.
Total vector size including the insert is around 10kb.
Could the lower transfection efficiency be directly caused by the missing 5' insert?! Or do you have any other suggestions, what could be the problem here?
Thanks for your input!
Katharina
Hi Katharina,
It seems you are putting lots of thought/effort into solving the potential problem of different transfection efficiencies of the two different plasmids in multiple cell lines. While size of the plasmid could definitely impact transfection efficiency (usually larger = less efficient), have you considered that maybe transfection efficiency is roughly equal and that instead, transcription, mRNA stability, or translation of the GFP is different depending on whether there is a cDNA coding sequence preceding the IRES sequence? This could also produce the results you are observing. I wonder if the GFP expressed from the plasmid lacking the 5' insert has a 5' methyl-cap. Nevertheless, a 10-15% difference in transfection efficiency is probably within a reasonable margin of error to proceed, unless of course you have an interesting finding regarding whatever gene you are inserting before the IRES sequence. If its impact on a biological process is 10-15%, then your attention to detail and concern with this issue makes lots of sense. Good luck.
Hej Martin,
Thank you for your answer! I was especially wondering whether any of the cellular processes coming after the transfection itself could be the reason here! Actually, I am trying to find out whether I would be better off with another vector system; since a stable signal coming from the GFP as a matter of transfection control is part of the work-flow at the moment... but maybe I should move away from that?! Hmmm... I am also a little bit concerned about the mediocre transfection efficiencies altogether, since they were also seen in easy-to-transfect cells...
Again, thanks for your input; and now that you say it, 10-15% really doesn't sound all that much, you're definitely right!
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