G418 advice in estab;lishing stable cell line - (Aug/02/2012 )

Hi,

I transfected CHO cell line for my gene of insert and I used G418 as selection marker(plasmid for which is seperate than plasmid for my gene of interest)

Transfection went well and I added G418 after 24 hours and I kept the cells for recovery. On 7th day I passaged cells but I forgot to add G418 and I realised it after 3 days(over the weekend). I passaged my cells and added G418 again at concentration of 550microG/mL.

Do you think I must have lost successfully transfected cells and the culture will over take with non-tranfected cells only now?
Or still can continue to recover my cells with constant selection pressure?

Please awaiting your reply.

Thanks
SBN

They will probably be fine, as G418 is a pretty strong selection agent. However, if I am reading your post correctly, the G418 is on a separate plasmid to your gene of interest, which makes it quite unlikely that your gene will be selected for, so don't be too surprised to find a very low number of integrants.