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.
Mark L. Huang, Ph.D. Zai4jian4! .
E-mail: rin0mxw at bumed30.med.navy.mil __l__ ______ .
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