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G418 selection of PA317

M. L. Huang rin0mxw at bumed30.med.navy.mil
Thu Jul 14 15:01:48 EST 1994

On 13 Jul 1994 10:46:40 +0100, 
Graham Atherton  <grggta at picr.cr.man.ac.uk> wrote:

>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.

We routinely use 750 ug/ml (ACTIVE conc; this varies from batch to batch) 
for both PE501 and PA317 cells. I agree that HAT preselection before 
transfection is key to get higher virus titre. G418 selection takes about 
a week to take effect.

************************************    Peace_Peace_Peace_Peace_Peace_Peace
Mark L. Huang, Ph.D.                              Zai4jian4!              .
E-mail: rin0mxw at bumed30.med.navy.mil           __l__   ______             .
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Immune Cell Biology Program                                               .
************************************ Bye! Au revoir! Ciao! Auf wiedersehen!
Opinions stated here are solely mine and are not those of the U. S. Navy.

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