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[andyg2886]

Hi

I have transfected two separate expression vectors into a human embryonic stem cell line. The first vector carries the puro-r gene, and the second vector carries neo-r.

After the first vector was transfected in, I treated all cells with 10ug/ml puromycin to select the cells. This worked fine. I then transfected the second vector in and treated with 150ug/ml G418. This too, seemed to work fine. All control cells (without resistance) died under these concentrations. Puro/G418 selections were performed for 1 week.

I've now come to use these cells in an experiment. My cells have been out of selection for about three weeks while they've been bulked up. So, I thought I'd select once again just before I used them, to be 110% sure of what I'm working with.

I combined puro and g418 together in the media. The next day, pretty much all my cells were dead.

I've been racking my brains as to why this has happened. In the literature I found this:

"6. Drug selection can begin 24 hours after electroporation. If you are selecting with G418, refeed the plates with fresh medium and the drug for 6-10 days. HAT selection is for 8 days, with 2 days of HT following, to release the cells from selection. Puro should be used for 4-6 days. G418 and Puro cannot be co-applied, as their methods of action are the same. FIAU (0.2μM) selection usually can proceed simultaneously."

So puro and G418 'CANNOT' be used together. What does that mean? That they won't work if combined, or that they'll kill the cells, as has happened to me?

If you have any ideas please let me know because I am baffled.

Cheers

[bob1]

Basically it means that the mechanism of killing is similar for both -blocking of translation.  This would mean that adding both could be lethal at 1/2 the concentrations you might expect.  This means that you can't co-select, but you should be able to select sequentially and then use both after this (in theory at least).

I suspect that you lost the plasmids/integrants during the bulking up period - you need to keep the cells under a maintenance selection while growing to ensure that the resistance gene is not methylated or deactivated in some fashion.