Tet Inducible Gene Expression

Edward Edward
Wed Nov 29 13:19:08 EST 1995


Subject: Tet Inducible Gene Expression
From: mit.edu, 
Date: 29 Nov 1995 15:11:59 GMT
In article <mit.edu-2911951015050001 at cancer-ctr-mac-7.mit.edu> mit.edu, 
writes:
>I am utilizing the Tet inducible (repressable) system for conditional
gene
>expression developed by Gossen and Bujard.  I've found that lines which
>initially showed inducible expression of the desired gene eventually
cease
>to induce these genes after several passages.  Anyone else seen these? 
>What causes this?  How do you prevent it?  Thanks
>
>Scott


	Apparently this loss of the tet repressor-VP16 fusion is due to the
long-term toxicity of this protein.  I've been told (by the inventor of
the new and improved version of this system) that the VP16 binds up other
transcription factors and "squelches" leading to slower cell growth. 
Several other lines have ceased expressing so this seems like a common
problem.  The new version of this system places the tet repressor-VP16
fusion under the control of its own target promotor thus making it
autoregulatory.  In the presence of tet, the tiny amount of leakiness
keeps a small amount of the transactivator around (without any apparent
detrimental effects).  With tet withdrawl, the small amount of fusion
protein binds its own promotor and amplifies (whoosh) and you generate a
terrific induction.  See the following paper for a more cogent
description:

Shockett et. al., PNAS 92(July) 6522-6526

	BRL now markets their kit (for a hefty $295) though it isn't in the
catalog yet.  If you're interested the catalog number is 10583011 and I
can tell you from personal experience that it is on backorder for several
weeks!
					Ed
Edward C. Goodwin Ph.D.				Yale University
ecg at po.cwru.edu					School of Medicine 
"Facts do not cease to exist because 		Department of Genetics
they are ignored"- Aldus Huxley			P.O. Box 208005
								New Haven CT   06520-8005



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