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Hygromycin selection of virally-transduced cell lines - (Oct/01/2012 )


I am developing stable cell lines that overexpress a gene of interest. The virus I have transduced with contains a hygro-resistance cassette. I made virus in 293T cells (transfected with two packaging viruses and the virus that contains my gene of interest), then added fresh, .45uM filtered medium containing virus to two carcinoma lines for 3 days straight. I then passaged the cells and began selection at 50ug/ml hygroB. One of the cell lines doesn't appear to be affected by Hygro, while the other is taking a while to pick back up because this line is just sensitive to trypsin and passaging in general.

For the line that hasn't really died off yet, can I simply change the medium and scale up to 100ug/ml or higher? Or, do I have to passage them AND increase the hygro concentration? How quickly should I start seeing cells die off?




What you should have done is to work out the optimal concentration for selection of the 293T with hygromycin before you started this experiment. Then you would have used a concentration that you know works...

Most of these sorts of drugs don't work well at high levels of confluence, you need the cells to be at or below 70%, but it works better at 50% or below.