We are trying to make stable transfectants in NIH 3T3 and 3T3-L1 cells. We are successful in getting our transgene (GFP-fusions) into the cells. However, after some time under selection (G418 mostly) we loose the expression of our GFP-fusion, but retain resistance to G418. Does anyone have suggestions how to avoid this problem?
Plasmid: CMV-promoter, G418 resistance - we do not liniarize, but this is one of the things we want to try next. Where in a plasmid relative to the transgene is the best position to liniarize?
Transfection: Lipofectamine 2000 or Lipofectamine PLUS
Selection: 500 µg/ml G418 medium added the day after transfection.
Morten Pręstegaard, Ph.D.