Methotrexate amplification?

Rob Jordan rjordan at u.washington.edu
Wed Aug 16 11:36:45 EST 2000


> In article <8n8jgh$oa4$1 at oyez.ccc.nottingham.ac.uk>, "Student"
> <mucineer at iname.com> wrote:
>
> > I have a question which is puzzling me. After transfection and selection
> > (with media lacking ribo/deoxyribonucleosides and G418) of a CHO cell
line
> > trasnfected with my plasmid of interest, it is possible to "amplify" the
> > copy number of the gene in the host chromosome by adding increasing
amounts
> > of methotrexate.

I'm not sure I understand. Methotrexate causes amplification of the
dihydrofolate reductase gene. Is this the gene on your plasmid? I tried
increasing the G418 concentration in some transfected cells that I had
(before cloning) to try to select for cells that had multiple copies of the
plasmid (and thus might be more resistant to the G418). We put that project
on hold for other reasons before I had a change to check it out, but it
doesn't seem to have been really successful.

> > Exactly how do these pieces of DNA which is already incorporated into
the
> > host chromosome "increase in number"?

Do a Medline search for G. M. Wahl, if you're really interested. He would be
a starting point at least.









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