Can multiple plasmids comete in transfections?

John Ladasky ladasky at my-deja.com
Sat Mar 16 00:32:58 EST 2002


Hello again,

A few days ago I posted that I was trying to make a cyan fluorescent
protein (CFP) fusion construct out of a yellow fluorescent protein
(YFP) fusion construct.  Well, I did it.

I'm using various CFP and YFP chimeras in fluorescence resonance
energy transfer (FRET) studies of protein interactions in vivo.  I
have returned to the microscope after a few weeks at the molecular
biology bench -- and I've rediscovered an old problem.

I am transfecting the human cell line HeLa.  I have performed the
double transfection in two ways: either as a transient transfection
with both the CFP and YFP plasmids simultaneously; or, by establishing
stable lines with the YFP plasmid, then transfecting these transiently
with CFP.  In both cases, I've observed an extremely annoying pattern:
those cells which are CFP-bright are YFP-dim, and vice versa.  This is
making it extremely hard for me to do FRET!  I do want variations in
the expression levels of the two proteins from cell to cell, but I
need them to be somewhat uncorrelated -- positive correlation I might
be able to work with, but negative correlation is a disaster.

All of my plasmids are built from Clontech's pN3 vector.  Therefore I
have the same eukaryotic promoter in each plasmid, the ever-popular
human cytomegalovirus immediate early promoter (pCMV-IE).  One of my
colleagues suggests that a transcription factor which binds pCMV-IE
may be in limited supply.  Thus, when I succeed in obtaining a high
level of expression from one promoter, it comes at the expense of the
other promoter.  This seems plausible to me.  Does anyone out there
know if such a phenomenon has been reported?  I'm combing Medline, but
as you might imagine, a keyword search with the terms "promoter",
"competition", and "transfection" is awfully broad...

If you can think of any other reasons why I might be seeing negative
correlation between the expression levels of the two proteins, that
would be useful as well.

Thanks again!

--
John J. Ladasky Jr., Ph.D.
Department of Biology
Johns Hopkins University
Baltimore MD 21218
USA
Earth




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