HEK293 WT showing resistance to G418 - Please, I need ideas and opinions (Nov/15/2006 )
I got two different batchs of HEK293 WT from other labs (not from a company) with which I would like to create a stable cell line soon.
I did G418 (Gibco) assay to see how long it take them to die. For my surprise after two weeks with selective medium (1mg/ml G418), they look quite happy. I had repeated this assay three times with identical results and I used this antibiotic in another cell line and they were death in a week more or less.
I heard that some HEK293 WT show resistance to this antibiotic. Is that true? Has someone come accross this type of issue in the past? Is there any reference or information in regards to this?
Anyone could give me some help or suggestions about what I'm doing wrong or what else I could try?
Someone who is currently lost.
PS: I think I'm going to buy a new batch from a company, but first I would like to know how this happens. (Isn't it unlikely that two unreleated different labs have given me this supposed HEK293WT showing this resistance?).
That is a high concentration that should kill them. I would be worried that the cells are contaminated with a resistant line. You could try to PCR out the neo resistance gene to check on this.
yep, abstract genomic (or total RNA to cDNA), detect the neoR gene's CDS.
Thanks for the advice. I already got the primers for Neomycin cassette and I will try that very soon. This is probably the most probable cause. However, do you think it could be due to a different cause, even an unlikely one?
Other question I have is how on earth the contaminated cells are become predominant? I can understand that some shows that resistant because the contamination issue but most of them is unlikely.
you need to divide more your cells as the neo is active only in active cells.
Sorry, I don't understand. What exactly do you mean with divide more? Do you mean that they should be less confluent? Aren't all live cells active?
I'm a little bit confused (as usual) ;-)
well you understood good.
Confluent cells tend to stop divide and thus are considered less sensible against G418.
i think also : check end of use date, and maybe the concentration of stock if it's not you who prepared it.
But since the G418 is working on the other cell line, it seems it is no a reagent or technique issue.
but i though that tumorous cells and especially hek293 and Helas were hard to select...
moreover the sesibility is cell type dependant...