Bernard Murray, PhD
spam at 127.0.0.1
Mon Aug 14 22:10:16 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.
> Exactly how do these pieces of DNA which is already incorporated into the
> host chromosome "increase in number"?
I don't know the exact mechanism (and would be fascinated to hear
any fact-based theories) but if I understand correctly it is thought
that the gene duplication occurs during DNA replication. The strong
selection pressure causes these rare (and presumably somewhat otherwise
impaired) cells to outgrow the others. Similar phenomena are seen
when selecting for amplification of MDR1 or CAD. I've seen the
inducibility used as an indicator for general genomic instability.
This does seem to be an in vitro-specific phenomenon as eg. there's
no sign of MDR1 amplification in vivo.
Bernard P. Murray, PhD
bpmurray at cgl . ucsf . edu
Department of Cellular & Molecular Pharmacology, UCSF
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