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Stable protein expression in Vero cells - (Sep/06/2012 )

I have been trying to generate Vero cells stably expressing a Golgi protein fused to GFP or a membrane protein fused to GFP. I started 3 months ago with this and until today I do not seem to have any positive colonies! I did different transfections and after 48h I split my cells in the presence of G418 (1mg/ml). My control cells they all die within 10 days but in my transfected cells I see little colonies after this time. When I check my cells in the fuorescence microscope some of them are positive but the colonies are completely heterogeneous. When I split again the cells I loose my few positive cells....
Any idea how to improve this?
Thank you very much!!


Is it not too much 1mg/ml of G418? Are you using the lower concentration to kill the controls? It sounds to me that you are using too much Neomycin and your cells are suffering...


Hi! Thank you for your answer. I did a killing curve on my Vero cells and 1mg/ml was the concentration recommended for mammalian cells and that killed all the control cells in around 10 days...when I used 0.5 or 0.8 I saw some cells still alive and growing in my control. But I could try a lower concentration in the transfected cells to see if I can get more positive cells from the beggining. Thanks!