Establishing stable cell lines...

Dima Klenchin klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Wed Sep 13 21:37:29 EST 2000


In article <20000914000236.25837.qmail at web3707.mail.yahoo.com>, jonny_jump at yahoo.com (Jump Jonny) wrote:
>Dear All
>
>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.

That might be a problem - you cannot assume G418 
concentration for a particular cell line beyond very rough 
guidelines. G418 is never pure and active content vary 
between manufacturers. Different clones of the same 
parental line frequently show different levels of 
resistance (I've seen CHOs that needed 800 ug/ml -
more like Jurcat than "normal CHOs).

You need to do a pilot and find minimal [G418] that 
kills all your non-transfected control in 14 days.

>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 ?  

That might ostensibly be a case but it would mean a much
bigger problem - you are not doing proper controls in your 
experiments! :-)

>Might a less efficient
>transfection protocol like calcium phosphate work
>better in the long run ?

I don't think so. 

- Dima






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