DHFR-mediated gene amplification
tsteenstrup at hotmail.com
Mon Feb 19 10:46:30 EST 2001
I don't know if the real mechanism behind it is known, but it does not
involve a replication origin. It is probably and endogenous recombination
that is taking place instead.
The whole point of doing this procedure is to amplify a gene of interest.
This is usually used in cases were you want to boost yields. The MTX
selection is only a tool. What would the point be of transfecting only with
a DHFR gene?
You can use DHFR+ cells, but you get better results with DHFR- lines.
Timeframe depends on how high you want to go and how many steps you want to
do. In my experience, each step in MTX concentration takes a couple of
weeks. More steps (may) give better amplification. It's a balance, where you
want to increase pressure to increase yield, but at the same time, you must
be careful to avoid deletion of your gene of interest.
A gene-IRES-DHFR construct will give the best result, as there's a better
correlation between yield and MTX pressure.
If you haven't read them, I would recommend that you read RJ Kaufmans papers
on the subject.
"Jens Tornoe" <jens at tornoe.net> wrote in message
news:3xgj6.164$uO5.179613731 at news.mobilixnet.dk...
> We are considering increasing transgene expression in our cell lines by
> of the DHFR/MTX system. I have been scanning the literature, but I could
> still need some advice from people with hands-on experience.
> I would highly appreciate any response to the following questions:
> 1) Is there any consensus about the mechanism of replication? Is there an
> origin of replication located within the DHFR sequence? Our construct
> supposedly contains only the cDNA of DHFR, which should then contain such
> origin. I have even read that one can achieve amplification of a desired
> gene by co-transfection of a DHFR plasmid, making it even harder for me to
> figure out the mechanism of amplification.
> 2) Can one use MTX selection for DHFR-amplified cells in a DHFR+ cell
> Apparently, a CHO/DHFR- line is most commonly used. We would like to use
> own DHFR+ cell lines, if possible.
> 3) What is the typical time-frame for obtaining 'boosted' clones?
> Thank you in advance for any response given!
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