Promoter competiton in pCMS-EGFP?

M.J.Schell mjs54 at cus.cam.ac.uk
Fri Sep 15 14:11:23 EST 2000


I have been using Clontech mammalian expression vector called
pCMS-EGFP.  This vector has a CMV promoter upstream of the MCS to drive
the cloned gene and it also has another promoter (SV40) that drives
eGFP.  Thus, cells expressing the vector should both be green and also
produce protein from the cloned cDNA.  If so, this would be great
because one could identify the green expressing cells while they are
still alive without having to do multiple-plasmid transfections.

However, when I transfect  cells and stain them with a good antibody to
my cloned protein, I find that only a subset of the green cells (perhaps
20%) stain for my protein (the staining is very strong).  These cells
appear only weakly eGFP-positive, while neighboring cells are strongly
green but not stained for my protein at all.  There are no cells which
make my protein but are not green.  How can this be?  Here's some of the
things people have suggested, but nobody seems to know for sure:

1.  The vector contains and intervening sequence (IVF) between the CMV
promoter and the MCS which, according to Clontech, "is an intron that
is efficiently spliced out following transcription".  Perhaps the cells
not expressing my protein didn't splice out the intron.

2.  "Promoter competition."  Nobody seems to know exactly what this is,
but I am imagining that if a fair bit of the cells translation machinery
is tied up making GFP, perhaps it can't also produce the cloned protein
transcribed from the other promoter, especially since eGFP is about 20
Kd and my protein is 50 kD.

3. The green cells not expressing have somehow "kicked out" my cDNA from
the vector.

I have queried Clontech about this but so far no reply.
Any ideas or suggestions would be appreciated.

--
  M.J. Schell
  Department of Pharmacology, University of Cambridge
  Tennis Court Road, Cambridge, UK CB2 1QJ
  +44 1223 339682/ <mjs54 at cam.ac.uk>








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