inducible eukaryotic expression

Annette vertino_bell at cber.cber.fda.gov
Thu Feb 29 16:14:12 EST 1996


Hi Folks.

I am interested in hearing others' experience with inducible eukaryotic
expression.

Specifically, my problem is this. I would like to over-express my protein
in CHO cells. I am able to express it transiently, but when I try to make
stably transfected lines, none of the selected drug-resistant lines [>25
screened] express my protein.  I am therefore working with the theory that
my protein must be toxic to the cells for lack of an alternative
explanation. Can anyone think of another explanation?

I would like to try one of the inducible systems to create a stable line
transfected with an inducible expression construct where the inducing
agent is (ideally) not likely to affect growth or membrane traffic.  The
LacSwitch (Stratagene) and Shockett-improved tet-regulatable systems look
intriguing, but my question is, has anyone successfully made the
double-stable in either of these two systems? I've heard rumors that no
one but the Stratagene people themselves have been able to make the double
stable in the Lac system. 

Also,  what are my options for single plasmid inducible expression? 
I'm afraid the use of glucocorticoids in the MMTV promoter system will
alter the growth and/or membrane traffic of CHO cells. What about the
metal induction of a metallothioneine promoter? Because my protein may be
toxic, and I don't know how toxic, I have to be concerned about
"leakiness" of the system as well.

Any and all advice/lab notebook excerpts/comments would appreciated.

Annette Vertino-Bell
<vertino_bell at cber.cber.fda.gov>

Annette
<vertino_bell at cber.cber.fda.gov>



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