CMV promoter-sequence and activity?

Stephen Klautky sklautky at vaxa.weeg.uiowa.edu
Fri Mar 20 04:56:00 EST 1992


In article <1992Mar18.094434.7318 at klaava.Helsinki.FI>
Tomi.Makela at helsinki.fi (Tomi Makela) writes:

>We have been using a CMV-B-gal construct as a control for transfection
>efficiencies in transient transfections. During these experiments we
>have noticed that this plasmid is not a very good control, because the
>CMV promoter itself is regulated to some extent by our other plasmids.

This is interesting.  Could you elaborate?
How is the CMV promoter regulated by other plasmids?
Do you know, for instance, if the promoters of the other plasmids are
involved in transcription factor competition?
Are you cotransfecting with plasmids containing cDNAs for transcription
factors that may affect the promoter?  

>Now we would be very interested in finding out the sequence of the CMV
>promoter, and we would also like to hear from other constructs used for
>monitoring transfection efficiencies.

We also use a CMV/b-gal plasmid.  It is described in MacGregor and Caskey
NAR 17:2365, 1989.  The title is _Construction of plasmids that express
E.coli b-galactosidase in mammalian cells_.  The CMV promoter used is the
human CMV immediate early promoter/enhancer.

I downloaded the sequence for the human CMV IE1 promoter a little while
ago.  I assume that part of this sequence is used in constructing
MacGregor's reporter plasmid.  The accession # is X03922.  In the database
the name is: Gb_vi:Hs5p1.

The title of the paper associated with the file states that the promoter
binds NF1 in five places.  In addition there are other known interactions
that might confound its use as a control.

We use CMV/b-gal for some of our cells while RSV/luciferase is useful
for others.

Unfortunately, luciferase is not a solution for all our problems.  It
is not stable in our primary cell extracts so we must be extra careful
about handling the extracts and thaw them in batches for assaying.  There
was a Biotechniques report sometime back on the preparation of
luciferase-containing extracts.

If you are testing promoters then you may be using CAT as your reporter.
I'll assume you have RSV/CAT etc.  You can buy other reporter gene
constructs such as ones containing the growth hormone cDNA.  This
protein is secreted into the media and is detected by an RIA.

There was also a Biotechniques report on performing an enzyme assay for
neo.  I don't know how sensitive it is compared to the others however.

There are probably others that I have missed.

Theoretically, using the HSV-TK promoter may avoid problems compared to
using other control promoters that contain enhancers.  Enhancers can be
targets for hormonal regulation and may be affected by cell growth etc.

-=] Steve [=-        SKLAUTKY at VAXA.WEEG.UIOWA.EDU



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