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G418 selection of PA317

Graham Atherton grggta at picr.cr.man.ac.uk
Wed Jul 13 04:46:40 EST 1994

Subj:	Re: PA317 and PG13 (mouse embryo) cell culture?

In article <Csoy3y.Ds at cerc.wvu.edu>, arnold at cs.wvu.edu (Greg Arnold) writes:
> 	Hi!  If anyone has worked with these cells, your input would be
> appreciated.  Specifically, I am trying to do a kill culture with these
> cells which are to be transfected with MLV-based retroviral vectors.
> The kill cultures are to check for G418(Neo) and Hygromycin resistance.
> 	In some of the literature I've seen, they've used G418 concentrations
> of 1.5-2.0mg/ml.  My advisor seems to think that is kind of high.
> So if you have any ideas/suggestions about culturing these cells, please
> e-mail me.
> 					Greg Arnold

>I use mouse embryonic stem cells; I have no idea if your lines behave anything 
>like ES cells, but I do know that our cells are sensitive to about 
>300-350ug/ml G418, and 150-200ug/ml Hygromycin (don't forget that at least 
>with G418 there is batch to batch variation in specific activity).  The high 
>G418 concs (above 1.5mg/ml) are necessary for lymphoid cells, which don't get 
>too worried by G418.  I'd suggest you plate out duplicate 6-well plate 
>cultures with a range of concs., from zero through to 500ug/ml in 100ug 
>increments (you can fine tune the range in a second experiment).  If you need 
>more than 500ug/ml G418 I'd be surprised, unless they are lymphoid cell lines.


>NNNN   NN  Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz)  ZZZZZZZ  
>NN NN  NN       Cytogenetic and Molecular Oncology Unit          ZZZ
>NN  NN NN           Christchurch School of Medicine            ZZZ
>NN   NNNN              Christchurch, New Zealand              ZZZZZZZ
	      Phone (64-3)364-0880  Fax (64-3)364-0750

We routinely use 1mg/ml G418 for 3T3 and like cells (we treat PA317 as similar).
This gives us good selection as long as the cell are sub-confluent when G418 is
added - cell division is essential for this drug to work. I agree with Martin
that empirical determination would be best. A word of warning on PA317 - viral
production is markedly enhanced in our hands if selection is carried out in
the presence of HAT. Stressing the cells by selection in G418 seems to make
the cells lose the viral packaging function.

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