Dear all,

Andre Hamel hamel at ccu.umanitoba.ca
Tue Mar 2 18:25:24 EST 1993


In article <BRIANF.93Mar2100454 at dna.uvm.edu> brianf at dna.uvm.edu (Brain Foley) writes:
>I do not have references handy, but I believe that the MMTV LTR
>requires glucocorticoid receptor for expression.  Not all cell lines
>express glucocorticoid receptors.
>
>From what I have read, the MMTV LTR is quite eficiently shut off in
>the absence of the glucocorticoid/glucocorticoid-receptor complex.
>The reason you see some expression before adding dexamethasone to your
>L-cells is that there are glucocorticoids in the media or serum that
>you are using.
>--
>********************************************************************
>*  Brian Foley               *     If we knew what we were doing   *
>*  Molecular Genetics Dept.  *     it wouldn't be called research  *
>*  University of Vermont     *                                     *
>********************************************************************


We've found it necessary to routinely treat our fetal calf serum with FOUR
SUCCESSIVE changes of activated charcoal (gently stirring, using about 10
grams per 500 mL serum), followed by sterile filtration.

This is done for all cell culture work involving MMTV LTR as well as
several more sensitive promoter/enhancer systems (ARE and GRE types).

Then, depending on cell line, expression vectors for GR or AR are
co-transfected with the enhancer/promoter/CAT or Luc fusion vectors.

Andre Hamel
Manitoba Vet. Virol.
Winnipeg, Manitoba
Canada

email: hamel at ccu.umanitoba.ca



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