NIH 3T3 Geneticin selection problem - (Jul/30/2010 )

Hi!

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,
Cheers,
Roland.

1.5 mg/ml is a huge concentration, recommended concentrations range from 50-1000 ug/ml. What you need to do is a titration of the effective dose and choose the minimum effective dose. Using too much leads to transfected cells that are resistant but are seriously damaged by the drugs and as such can not be easily compared to the untreated cell line.

bob1 on Sun Aug 1 22:09:29 2010 said:

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,
Roland.

Non-viable cells (as opposed to dead) aren't necessarily rounded up and/or floating, they could be senescent for instance.

I'm having a similar problem. I used Geneticin at 500 ug/ml and did my clonal expansion from a 96-well. They reached 90% confluency in a T-25, and I decided to expand them into five T-25s for freezing down. Now only a handful of cells are fluorescent. What's going on?

Did you keep them under a maintenance level of selection? Inserted plasmids are often methylated heavily, and can be methylated such that the gene of interest is methylated, but the resistance marker isn't. Sometimes fusion products are toxic to the cell, so it can't easily produce the product without dying.

Hi Bob, thanks for your reply.

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?

Your best bet would be to assay for your gene of interest and see if they are still expressing. Unfortunately I don't think there is a solution to the methylation problem.