Stable transfection of humanFibroblasts - (Nov/06/2001 )

I have tried, for over a year now, to obtain stable transfectants with MRC-5 cells (primary human lung fibroblasts). Thus far, we have obtained some clones that survive despite selective pressure (400 µg/ml of G418). At first, cells have divided slowly but consistently. However, after a few population doublings, there appear to be no further cell division although the remaining cells appear to be in good shape. Would anyone have suggestions? Would it help to increase FBS concentration or to lower the selective pressure? Right now, I cannot afford to loose these cells!

Hi. We are also interested in starting stable-transfected, primary dermal fibroblast cell lines. Do you have any advice on how long before you were able to achieve population doublings? We have been working with G418 doses at 50ug/mL and there growth is very slow if at all. Thanks.