NIH 3T3 Geneticin selection problem
Posted 30 July 2010 - 03:44 AM
This is my first post, so please excuse any deviations from the norms.
I am trying to establish stably expressing cell lines (polycloncal populations to start with) in NIH 3T3 cells. My problem is that I have a lot of non positive cells growing, which simply refuse to die even at very high concentrations of geneticin. I am assuming these cells are negative for my construct as they are not fluorescent. The selection has been done with either 1.5 mg/ml or 2.0 mg/ml geneticin in DMEM + 10% FBS and penstrep, as well as 30 mM HEPES. After an initial and quite massive cell death (something like 60 percent), I get formation of foci (not clonal) which I think could be due to higher resistance of confluent cells to geneticin. These keep growing at the edges, and eventually lead to a fully confluent flask.
I am using Geneticin from GIBCO, stock active concentration 50 mg/ml. I have seen various concentrations used for selection of NIH 3T3's in litterature - I chose very high concentrations based both on this and previous experiments where the cells also did not die off as expected. I have never tried using less than 1.5 mg/ml - could it be that I am using too much, and this somehow leads to development of resistance?
I would be very thankful for any help or comments,
Posted 01 August 2010 - 02:09 PM
Posted 01 August 2010 - 02:45 PM
Thanks for the reply - I will try lower concentrations (I have done kill curves, and at 500 ug/ml the cells are essentially unaffected after three days, whereas at 1 mg/ml more than 90% are dead). Just out of curiosity, what's your opinion on the reason that I have so many surviving non-positive cells (as judged by fluorescence) at these high concentrations? The expected result would be that all cells would be dead if the drug concentration would be too high, and I find it counterintuitive to think that a too high concentration could lead to higher survival, at least in the this time interval.
Many thanks for your reply,
Posted 02 August 2010 - 01:27 PM
Posted 05 September 2012 - 01:02 PM
Posted 05 September 2012 - 01:32 PM
Posted 05 September 2012 - 10:30 PM
It was under 200 ug/ul maintenance selection during expansion. I have upped it to 500. Can you please explain a little bit about the effects of methylation?
Are the non-fluorescent cells only expressing the resistance gene and not my gene of interest? How shall I handle this?
Posted 06 September 2012 - 12:29 AM