G418 and C6 Glioma Death / Stable C6 Line - (Nov/02/2009 )

Hello all,
I am attempting to create a stable cell line with a GFP gene inside a pcDNA 3 vector. Therefore I have two strategies: G418 resistance or FACS. Unfortunately I have tried both with little success.

With G418:
My problem here is creating the killing curve. I've tried concentrations from 100ug/mL to 4mg/mL; above 800 ug/mL I see some cell death and slowing down of cell growth (their media takes longer to change color) but ultimately I never get "massive cell death." At best the cells stop growing or reach confluency. Obviously if they don't die, I can't select for resistant. I've been accounting for G418 only being 70% active and been dissolving it in serum-free DMEM, pH adjusted with NaOH.

With FACs:
I've tried sorting cells both clonally into 96 well plates and in a bulk container with many cells. In the case of 96 well plates, only 3-4 of the ~400 wells I plated grew cells and none of them had my GFP. I've been putting them in DMEM with 30% FBS, and P/S. In the case of the non-clonal cells, I see some retention of GFP, but after a few days it rapidly goes away and I'm left with normal cells. I believe my gene may be interfering with the cell cycle. To test this, I wanted to put my gene in a tet-on plasmid, but in order to do this, I need to do a double-stable transfection - the first step with G418.

So the real question is - why won't my cells die when I give them ridiculously high doses of G418? I was even paranoid enough that maybe these cells somehow acquired resistance in the past since I did not know their history. I ordered some brand new C6 cells from ATTC and there is no difference. Has anyone had experience treating C6 cells with G418 or can think of another way to do a stable transfection (or tet-on?) Thanks!

Cheers,
Andy

When you say that the cells stop growing... do you mean that they will start growing again if you remove the G418 or are they senescent?

If senescent, it is irreversible, so that would be appropriate for selection.

How long are you using for selection? If it is a few days, give it a couple of weeks... and make sure you seed the cells at low density, the selection doesn't work when approaching confluence.

Glioma are tricky to culture properly, the cells are large and have huge processes which means that they can reach confluency (and they will stop growing) at about 40-60% apparent surface area coverage.