Inducible Mammalian Vectors

[Victor Levenson]

Clay,

I have some experience with Tet systems, so this may be of interest.

1. Inducibility. Tet system is rather inducible - 100 to 300 fold;
there are reports about 500 fold but I have never seen anything like
that in my experiments. A friend who uses lac system claims that this
level of induction is what he is getting as well. Keep in mind that in
different cell lines this may be different.

2. Leakiness. Tet system is relatively leaky, both in ON and OFF
configurations. Two modifications with good inducibility and no
leakinness were reported in 1999: Helen Blau and W. Hillen. Both rely
on tetTA for induction and TR for repression; the problem is that
you've got to have different dimerization domains for these two
proteins and have to select a clone that expresses them in appropriate
ratio. However, you've got to determine tolearble leakinness: is it
absolute (one molecule will kill the cell) or relative.  

3. Kinetics of regulation. This is an important issue to determine
before you make your choice: addition of inducer can take more time to
take effect than removal (e.g. original Bujard's paper).

BTW, besides ecdysone, Tet and Lac systems there is another one, based
on mifepristone-induced activation.

I work with Tet system, so if you need practical advice drop me a
note.

Good luck,

Victor Levenson



On 23 Nov 1999 13:12:21 GMT, comstock at plains.NoDak.edu (Clay Comstock)
wrote:

>Hello;
>
>I was curious as to if anyone had any experience with either the Ecdysone,
>Tetracycline, Lac, or any other inducible vector.
>
>My main intrest is to study the effect of phosphorylation on the
>degradation of a histidine tagged protein in NIH3t3 cell culture. It seems
>to me that one could use these inducible vectors much like a radioactive
>pulse chase. For example, one could induce expression for 1hr then remove
>the induction agent and track your protein of intrest.
>
>If anyone could provide any insight as to the induction levels of any of
>these systems one might expect, how leaky are these systems, or any other
>information that might help me decide which system is best for me, it
>would be greatly appreciated.
>
>Thanks 
>Clay
>
>
>
>--
>~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
>Comstock
>e-mail:    comstock at plains.nodak.edu 
>Homepage:  HTTP://www.med.und.nodak.edu/depts/biochem/clay/comstock.htm
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