Inducible Mammalian Vectors

Nick Theodorakis nicholas_theodorakis at urmc.rochester.edu
Tue Nov 23 10:18:12 EST 1999


In article <81e3rl$kk5$1 at news.ndsu.nodak.edu>,
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

I once tried a similar strategy to study RNA degradation using the
LacSwitch system from Stratagene, and it failed miserably. I got great
induction of the RNA all right, more than ten fold, but the shut-off
after removal of IPTG didn't appear to work at all.

In terms of leakiness, I think all systems are leaky to some degree,
and the definition of how leaky something is depends on your
perspective on what level of expression you can tolerate, and whether
you care about absolute levels of expresion or -fold induction.

I haven't messed with the tet systems, but maybe the tet-off (if that
is the one that shuts _off_ after adding tet, I might have the
terminology wrong) might be worth a try.

Nick





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