Can multiple plasmids comete in transfections?
ladasky at my-deja.com
Sat Mar 16 00:32:58 EST 2002
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.
John J. Ladasky Jr., Ph.D.
Department of Biology
Johns Hopkins University
Baltimore MD 21218
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