loss of GFP upon selection - (Feb/22/2007 )
Hi everyone. I have a problem and I could really use your help.
I transfected MCF-7 and T47D cells with the pEGF-C2 vector using Lipofectamine 2000. The next day I had many fluorescent cells. I waited 48 hrs and then began G418 selection using 350 ug/ml. I checked before selection and there were still many fluorescing cells. After only 5 days of selection, I seem to lose all the fluorescent cells. 
I need to make stables for my experiments. Does anyone know how to fix this? Thank you so much.
I transfected MCF-7 and T47D cells with the pEGF-C2 vector using Lipofectamine 2000. The next day I had many fluorescent cells. I waited 48 hrs and then began G418 selection using 350 ug/ml. I checked before selection and there were still many fluorescing cells. After only 5 days of selection, I seem to lose all the fluorescent cells.
I need to make stables for my experiments. Does anyone know how to fix this? Thank you so much.
was the mock pEGFP or with a gene for a GFP-fusion protein expressed? expression depends at least on importance or toxicity for the cell
Both the mock and the GFP tagged protein show low expression
I have done stable transfection on 293 with EGFP and found the stable line gave much weaker intensity than the transiently transfected cells. EGFP from jellyfish can be toxic to cells. I think clontech, strategene or promega (I dont remember which one) now sells a better GFP construct with low toxicityfrom another organism. Maybe you can look into it.
have you titrated your G418 to find the correct concentration to kill your particular cell line?? if you are using a conc. of G418 that is too low, the non-fluorescent untransfected cells will not be killed and overgrow your culture.