I'm treating my cells with a drug inducing SSB rapidly converting into DSB. While I'm able to measure total level of DNA damage via Comet assay and all kinds of yH2AX, is there a specific DSB DNA repair assay? I know there are kits for BER and NER, but not aware of other types. Thanks
Hi Nina,
I also work a lot with primary human cancer cells or PDX and DNA damage repair. A quite fast experiment, using the same techniques that you have already used, might be to induce damage and check for resolution-repair of the induced breaks. You can check via comet assay or yh2ax foci detection at different time point after removing the drug. Other experiments that are more informative but require some optimization are the GFP reporter assay for Homologous recombination or In vitro NHEJ assay. Do you work with primary cells or established cell lines? Can you transfect your cells?
Please feel free to reach out for more details and help for your experiment!
Marcello
Hi Marcello, thank you for your advice. I've already done foci resolution and pulse-chase-recovery comet assays, I just wondered if there is something more specific. With resolution-repair I have a concern that the drug-resistant cells might proliferate after the drug withdrawal and therefore the 'repair' I see is not actually repair but new 'undamaged' cells. I will look into the reporter assays, thank you. Any particular reporter assays you could recommend?
Hi, you can do short time points, in few hours breaks should be repaired (if their number is not so high) and you can control for cell proliferation. If you know how fast your resistant and sensitive cells proliferate, you can choose the best time points for your experiment. I use a modified version of DR-GFP reporter assay from Maria Jasin lab for HR assay. For NHEJ I know that its possible to put cell extract together with two oligos and assay the annealing efficiency; however I havent done myself and I know it requires some optimization. Let me know if you need more help, we actually work in the same city.
Thank you Marcello, I will read about it and will let you know if I'd need more help!
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