Establishing stable cell lines...

Gauthier , Eric EGauthier at neorcc.on.ca
Thu Sep 14 07:58:54 EST 2000


I am trying to establish stable mammalian cell lines 
expressing a transfected gene by G418 selection in CHO 
cells. 
I treat the transfected cells with G418 @ 400ug/ml (I 
assume this is this enough ?) but don't see extensive 
cell death or gain much expression after 2 weeks 
selection. 

A few things come to mind: 

1. In order to ensure that only transfected cells are selected, you should
perform a dose-response experiment  with G-418. Try to cover a range of
G-418 that extends from 50 to 1500 ug/ml (this can be easily done in 96-well
plate and using a spectrophotometric method- like the MTT assay- to measure
cell viability). Some cell lines require much more than 400ug/ml G-418 for
effective selection: if you don't get this one right, you will never get
your clones...

2. Cell death with G-418 is usually complete after 1 week or so.

Could the problem be that I use Lipofectamine 2000 and 
get so much DNA into the cells that too many of them 
remain resistant to the G418 ? Might a less efficient 
transfection protocol like calcium phosphate work 
better in the long run ? 

The transfection procedure has nothing to do with the selection process
itself.

Cheers!

Eric R. Gauthier, Ph.D.
Visiting Scientist, Tumor Biology Group
Northeastern Ontario Regional Cancer Center
Associate Professor of Biochemistry
Department of Chemistry-Biochemistry
Laurentian University
Sudbury, Ontario
email: egauthier at neorcc.on.ca


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