Methotrexate amplification

Martin Offterdinger martin.offterdinger at akh-wien.ac.at
Wed Aug 16 07:50:06 EST 2000


Student <mucineer at iname.com> schrieb in im Newsbeitrag:
8nba05$cos$1 at oyez.ccc.nottingham.ac.uk...
> Dear All
>
> 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"?
>
> Any insights will be appreciated. Thanks.
>
This is a well documented system;
These are the basics
1) Your parental CHO cell line is dihydrofolatereductase-deficient (DHFR-)
and can therefore not make nucleosides
2) On your plasmid with you gene of interest you have the DHFR gene present
3)MTX is an DHFR-inhibitor (competitive!)
4) By using MTX the medium you force the cells to produce even more DHFR
(and to coampflify your gene of interest)
5) Mechanism: This is due to gene amplification. (Selective DNA
reduplication on the region of the chromosome where your plasmid has been
integrated upon selection)

You should also check out the literature on this; it is used for several
years already!!!

Martin







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