Help! temperature dependent transient transfection problem

John Storring STORR96 at MEDCOR.MCGILL.CA
Thu Oct 31 13:09:41 EST 1996


Hi all, got an odd prbolem for you:

I am working with an embryonic neuronal cell line transformed with a
temperature sensitive SV-40 T Ag. They proliferate at 33 deg and
differentiate to a more neuronal phenotype at 39 deg. When I do the
temperature shift to 39 I switch media from a 10%fcs in neurobasal (GIBCO)
to 1%fcs in 50/50 N2.1/F-12. 

I transiently transfect these cells with reporter gene constructs useing
Promega's pGL-3 (luciferase) reporter and a beta-gal driven by CMV
promoter. Transfections are done using PFx-7 (a lipid available from
invitrogen) and are done in the 33 degree incubator and followed with a
10% serum recovery for 8 h before I begin the differentiation. Harvesting
for enzyme assay is done 48h later. 

The problem is that cells which are shifted to 39 deg fail to express
either luciferase or beta-gal. This is true regardless of the promoter
being used to drive luciferase (either my promoter of study or viral
promoters (SV-40, SV-40 plus some enhancers)). Also the CMV-beta gal is
DOA. Cells maintained at 33 degrees show excellent enzyme activity.

I know from RNA studies that the message from my promoter does go up when
these cells are differentiated, so the promoter isn't shutting down at 39
deg.

So what's the story? Anyone got any ideas?
 
Some things I've considered (but have yet to test include):

1. temperature shift is destabilizing the reporter enzymes and they're
getting degraded rapidly.

2. the lower serum in the 39 deg conditions might be affecting transcription

3. For some reason the differentiating cells are "spitting out" my
plasmids at 39 degrees

This is a critical series of experiments for my work, and I'd appreciated
any thoughts, comments, experiences that anyone has to offer.

Thanks much,
John
J STORRING
STORR96 at MECOR.MCGILL.CA



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