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G418 mammalian cell selection question

cell culture G418 CMV

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3 replies to this topic

#1 watsonkatherine

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Posted 15 July 2013 - 02:48 PM

I am using G418 400-600ug/mL to select for my transfected cells. I have used fibroblasts, epithelial and endothelial cells. I had a general question - I am seeing both my transfected (GFP tagged) cells as well as my untransfected cells dying as well as surviving from the selection treatment. I have been selecting for 14 days now.

My question is how are my uninfected mammalian cells able to survive the G418 treatment if mammalian cell do not normally carry the neo-resistant gene? My plasmids that I transfected with do contain this gene.

#2 sssss

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Posted 15 July 2013 - 09:24 PM

Hi,
I have also started working with G418 selection in adherent as well as suspension cells. Are the cells cross contaminated. Have you ever checked the GFP positivity in untransfected cells after this problem. Prepare fresh G418 and check if it works.

#3 leelee

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Posted 15 July 2013 - 11:00 PM

My question is how are my uninfected mammalian cells able to survive the G418 treatment if mammalian cell do not normally carry the neo-resistant gene? My plasmids that I transfected with do contain this gene.


You are not using a high enough concentration of G418, if your untreated cells are surviving. You need to titrate your G418 stock and find the concentration required to kill your cell line. Then use this concentration for selection of your transfected cells.

#4 pcrman

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Posted 18 July 2013 - 09:06 PM

Different cell types have different LD50 and that is why for a particular cell line, you have to do a killing curve.





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