Dexamethasone-inducible pMAMneo vector

Bernard Murray bernard at elsie.nci.nih.gov
Fri Feb 28 17:08:43 EST 1997


In article <5f6ugs$3ij$1 at merkurius.lu.se>, Marit.Anthonsen at medkem.lu.se 
says...
>
>I have performed stable transfections in CHO-cells using the pMAMneo
>mammalian expression vector from Clontech. The gene expression should be
>dexamethasone-inducible. Unfortunately, we have problems. Tthat is: the
>induction has worked ONCE (for 37 clones) - 4 unsuccesful tries. I am
>wondering if anyone has experience with this vector, and if there is a
>"trick" (eg. in treatment of the dexamethasone?)

Can you expand on what your results are so far?  If you are not seeing
any transcription at all then it might be worth checking that the
glucocorticoid receptor is present and active - eg. blot for TAT after
dexamethasone treatment.  If the cDNA you have introduced is toxic
you may have selected for non-expressing cells.  You can over express
the GR if you ask Keith Yamamoto at UCSF as he has an RSV driven
version.
	The usual problem is activity without inducibility.  I hope
that you are using charcoal stripped FCS (FCS is full of glucocorticoids)
and if you are feeling extra paranoid you may want to use phenol red
free medium (although phenol red is more of an oestrogen receptor
agonist than a GR agonist).  Try a range of doses of dex or you may
be killing the cells.
	Overall the MMTV promoter can be quite leaky so you may want
to try vectors based on tandem GRE repeats.  Amersham/USB used to sell
expression vectors called pGRE5-2 that contained heptamerised GRE
sequences and were supposedly less leaky than the full MMTV promoter.
I don't see them in the current Amersham catalogue so I don't know
where you can get them now.
	Bernard

Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov  (National Cancer Institute, NIH, Bethesda MD, USA)




More information about the Methods mailing list