inducible eukaryotic expression

Robert Slany rslany at leland.stanford.edu
Fri Mar 1 11:58:34 EST 1996


Hi Anette
	seems we both have pretty much the same problems. I tried to make 
stables with my protein in 4 different cellines under the control of two 
different constitutive promoters and the inducible metallothionine system. 
None of them worked (that sucks!). I could even demonstrate that the plasmids 
toghether with my construct are present in the DNA (by PCR) and still the 
cells wont express the protein. 
The problem is that the metallothionine system is quite leaky in most 
cellines. In cos cell for example it is almost non-inducible and always 
switched on. In lymphocytes it seems to be more tight (I am using the sheep 
metal response element). 
The tet-system worked in a transient assay in cos cells for me and so I 
tried to make the double stables. To make stables with the tetracycline 
repressor/transactivator wasn't that difficult but since there are no good 
antibodies available you cannot test for expression easily. I did RT-PCR on 
my subclones. The only way to realy assay for tTA expression is to 
cotransfect with a reporterplasmid under the tTA control e.g. a luciferase 
gene (I did not yet do that). 
Unfortunately I haven't had success with the second stable transfection. By 
asking around I have found 5 (!) different labs that tried to make double 
stables and for NONE of them it worked (!). So I guess mine and your chances 
are pretty low. 
Now I am thinking of transiently transfecting my cellpopulation and sorting 
out the transfected cells by cotransfection with a single chain antibody that 
allows binding of the transfected cells to magnetic beads covered with the 
corresponding hapten. That is a commercial system sold as pHOOK-1 system by 
Invitrogen. I haven't done the experiments yet and therefore I cannot tell 
you if it works but it might be worth considering it in your case too.
If somebody could come up with a more easy system for eucaryotic 
inducible expression I am sure it would be a big success but until then it's 
a pure lottery (and tons of frustration).
If possible try to stay away from stables.
That's all I can tell you. I know that's not much of a help but maybe it 
saves you a lot of disappointment. Maybe someone else on the net has a 
success story and can give us some hints.

robert

-- 
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* Robert Slany, Ph.D.	    				       *
* Stanford School of Medicine, L216			       *
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* rslany at leland.stanford.edu				       *
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