tet- free serum

Danny Altschuler dla at galactose.mc.duke.edu
Tue Dec 3 18:17:43 EST 1996


Bernard Murray (bernard at elsie.nci.nih.gov) wrote:
: In article <581fsa$u1v at netnews.upenn.edu>, rcowherd at mail.med.upenn.edu says...

: I am intrigued by the need for tet-free serum which I assume is to be
: used to culture cells transfected with tet-controlled vectors.

: The antibiotic activity of tetracycline in solution is fairly unstable at
: 4degC and rapidly disappears at 37degC (certainly in bacterial media)
: so is tet stabilised in serum?  I would have thought the opposite was
: more likely as tet would bind to serum proteins.  Alternatively,
: can "breakdown products" of tet bind to the tet repressor proteins?
: Transcriptional changes are reported to start to occur as low as 10 ng/ml
: doxycycline (eg. 20 pM) so this would need 200 pM for media containing
: 10% serum.  Is it possible to add Mg2+/Ca2+  to the medium to chelate
: trace amounts of tetracycline?  How about standard heat treatment of
: serum - would that be enough to inactivate contaminating tetracycline?

: Just wondering if the fuss is justified...

: 		Bernard

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

  Hi, everybody. i'm trying the Tet system with negative results so far.
I'm using a retroviral system developed in Reeves Lab (J Virol ?).
Packaging cell line and infection seems to work fine as evidenced by a 
Bgal retroviral (non tet regulated). With the tet vectors I can select
resistant colonies but when I do -tet to check for expression I do not get
anything (not even for the luc positive constructs). I contacted Bujard's
Lab in Germany and they told me that several of the commercial available
sources of serum contain a tet or tet-derived factor that is able to
repress transcription. This is why I have posted the question . If this
make sense or not, I do not know yet but I thought would be easy to check
with a tested tet-free serum batch and then see.
So, I want to order the Clontech stuff. Unfortunately I've called today
and they could not tell me anything.
Does anyone know catalog number and price?

Thanks again to all the people that answeredf
Danny




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