stable transfections using SCC 9 cells - (Jul/26/2007 )

Hi,

how to make stable tranfections using SCC- 9 cells. i am transfecting these cells with gfp tagged Rac 1 plasmids and selecting clones with hygromycin . but all my cells are dieing after 2 weeks. i am not getting any clones. i am using lipofectamine for transfection. can any one suggest good protocol for stable transfection please

-ravuri-

What was the estimated % EGFP positive cells before selection? Have you done a kill curve on these cells with hygromycin and used right concentration of it?

-genehunter-1-

QUOTE (genehunter-1 @ Jul 26 2007, 03:10 AM)

What was the estimated % EGFP positive cells before selection? Have you done a kill curve on these cells with hygromycin and used right concentration of it?

hi actually i am not seeing any green colour cells and i am using 50micrograms for ml hygromycin. this is new for me.

-ravuri-

Well, either your construct or your transfection could be problematic. You have to see some green cells if you expect to get any stable clones, because you are using a GFP fusion construct.

-genehunter-1-

QUOTE (genehunter-1 @ Jul 27 2007, 03:25 AM)

Well, either your construct or your transfection could be problematic. You have to see some green cells if you expect to get any stable clones, because you are using a GFP fusion construct.

hi thank you for your reply. actually i am seeing some green colour cells after transfection but after 1 or 2 weeks all cells are dieing. ui tried to reduce hygromycin but no use. actually i am using a gfp construct with Rac1 protein dominant and constitutive active verstions. and i also used amaxa for transfection. can u suggest any better protocol . it will be very helpful for me because i tried so many times and still trying but no results.

-ravuri-

The process of establishing stable cell line is tedious.
Have you determined killing curve for hygromycin before your selection on non-transfected cells? i.e. Are you using the right concentration, not too high? This should be the first step to do.
Some cells dont replicate well when they are seeded in low cell density. They go through a differeciation state and quite dividing. I had trouble before with one of the scc cell line (scc27, cant remember now). I dont know if that may also be a fector in your case. I dont have a solution to this, maybe try to seed them in higher density before selection? But I know in some cases, cells are hard to kill if they grow at high density.

If you have failed everything, lentiviral vector maybe the only way to go. Good luck.

-genehunter-1-