selection of transfected cells - (Jan/04/2009 )
I transduced my suspension cells with retrovirus-neomycin and selected with G418 for two weeks. After that, I want to add some drugs to check the function of my target gene. I want to know if it is necessary to select the mono clony from my cells and how to do it. Limited dilution or something else? After that is it necessay for me to add G418 in the culture medium? Because I thought that G418 might have some effects on my drugs.
Hi, first thing, if you want to have a single rate of expression you should obtain monoclonal cells. To do this, you can try limited dilution or use cloning rings, I have used them for corning glass. They are glass cylinders that allow you to dettach one single colony.
The second thing is that from my experience two weeks of selection is not enough, and that you should be quite sure you have stable transfection before remove the G418.