CAT assay reproducability-response
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CBS%UK.AC.MANCHESTER-COMPUTING-CENTRE.NESSIE::CA.ON.SICKKIDS.RI.RESUNIX::JHU
28-MAY-1993 14:32:05.71
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From: j...@ca.on.sickkids.ri.resunix
Subject: Re: CAT assays-reproducability
>Dear All,
> Are there any old fashioned CAT assayers out there?
> I am engaged on a project in which I am attempting to analyse various
>putative enhancer elements for response to oncogenes (eg src, int, hst etc).
>We are currently using a reporter system based on the HSVtk promoter ie
>pBLCAT2. The big problem has been reproducability of the response. I seem to be
>able to get everthing from 30-fold induction down to nothing from experiment
>to experiment. Is this level of variation unusual? If so, any ideas on
>minimising it?
> Another problem is that the negative control ie pBLCAT2 containing no added
>enhancer, appears to respond up to 10x to oncogene transactivation, minimising
>any putative enhancer effects seen on the elements under test.
> Several other groups use this reporter system with no such apparent problems.
> We have tried controlling many perceived sources of variation with little
>lasting success, I can go into more detail with any respondents.
> HELP
>
>Graham Atherton
Hi Graham:
I do not know exactly what you are doing. But I think if you use
an internal control such as a GUS fusion, you may get what you want.
Good luck.
Jim Hu
Thanks Jim,
I assume that you mean the variation between experiments in my work
is due to fluctuations in DNA uptake. The difficulty in introducing an
internal control is that the internal control promoter can respond to
the cotransfected oncogene in my experiments, making within experiment
variation difficult to control but presumably not between experiment
variation, as long as I am comparing like with like. Do you know if
any GUS fusion constructs exist which may be insensitive to oncogenes src
int, hst?
I did not suspect uptake to be the problem as for no apparent difference in
basal levels of expression from the reporter(ie in the absence of the
transactivating oncogene) does not change noticably in an experiment which
shows a large response compared with one that shows little response. If
DNA uptake were to blame, the former would be higher than the latter?
Throwing out a wider question, where does the variation in DNA uptake as
assessed by transient transfection of reporter constructs come from? My
guess is changes in the growth state of the recipient cells, or differences
in the composition of the CaPO4 ppt caused by temperature variation between
experiments as the ppt is formed?