Inducible eukaryotic expression vector ?
s.d.huen at bham.ac.uk
Wed Jun 8 05:50:07 EST 1994
In article <1994Jun7.234147.1855 at alw.nih.gov>, bernard at elsie.nci.nih.gov
(Bernard Murray) wrote:
> In article <2sv5p6$7dp at pobox.csc.fi>, airenne at convex.csc.FI (Kari Airenne) writes:
> > Dear colleagues/netters
> > I am wondering if any of you have used LacSwitch Inducible Mammalian expressionsystem sold by Stratagene for protein production in eukaryotic cells. As far
> > as I have understood right there are only limited number of possibilities for
> > inducible protein production in eukaryotic cells and, at least for me, this
> > seems to be a good choice for that. So if you have used this system or if you
> > know any other good tightly regulated eukaryotic vectors, please let me know.
> > Kari Airenne
> There are many useful non-commercial offerings - Wurm et al's heat
> shock promoter (PNAS, 1986), McCormick et al's interferon promoter (MCB 1984),
> Gossen and co's tetracycline response (PNAS 1992), Richard Hanson's PEPCK
We've (ie. my colleague) used this in transient and stable transfections in
this lab. The regulation is very good in transient transfections, giving 3
logs upregulation on removing tet. Basal is low in this case (with
monoclonal against the target protein, it can be undetectable!). On stable
transfections, the basal can be higher (neighbouring chromatin?) and it can
be difficult generating lines expressing the tet regulator (toxic?) but
when successful, it works well enough for experimental purposes. My
colleague may have now implemented the double tet regulation as well (tet
regulated expression of the tet transactivator + tet-regulated expression
of target gene). Haven't tried it in transgenics but at least tet is stable
> (JBC 1994). Basically, any existing inducible promoter could be a candidate
> [ANYONE WANT TO OFFER ANY!]. The "classic", metallothionein, has the major
> problem that it is too leaky in the "uninduced" state.
The degree of control with the natural or a synthetic metallothionein
promoter also depends on the gene you express. In some cases in this lab,
it does very nicely thank you (eg. EBV LMP-2A gene). With the mMT-I
promoter, there are a set of acute phase response sites upstream which if
you express a gene involving these functions, will drive this promoter to
very high constitutive expression.
David Huen, Institute of Cancer Studies, Birmingham, U.K.
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