I established chemoresistant cell line increasing the chemotherapeutic agent concentration gradually. Then this cell line was knocked out with the help of CRISPR system. I need to perform puromycin selection. While 1.5 ug/mL puromycin could kill parental cells (which were not treated with drug), even 5 ug/mL puromycin was not enough to kill the transfected cells (drug treated and then knocked out cells). I do not want to damage the cells increasing the puromycin concentration. Should I increase the puromycin concentration or try any other selection method?
Thank you.
Hi,
what was the chemotherapeutic you raised resistancy against? Maybe the pathways are somehow related? And did you try how the non-transfected resistent cells react to puromycin?
Best,
Cindy
In that case, I would titrate the puro concentration for the resistant cells again. Seems like they behave differently.
http://www.ncbi.nlm.nih.gov/pubmed/2860971
http://www.ncbi.nlm.nih.gov/pubmed/2861892
There is evidence of such kind of co-resistance. So I wouldn't be surprised.
Sorry to say but you have described your problem in a very cryptic manner... and based on nearly "sign language" communication between you and Cindy, my guess is that have you have checked YOUR puromycin solution is still an effective killer to "any cell"......forget about cell you want to use for your study.... BUT any other cell..... if not then buy a readymade solution from Sigma or any other vendor...... moreover the puromycin concentration for selection ranges from 0.5ug/ml to 10g/ml without being toxic to cells (which have acquired a puromycin resistant gene inserted in their genome.....) the next assumption is that you are using some kind of retrovirus or lentivirus vector to transfer the gene or some RNAi to your cell and not simply transfecting the cells with puromycin plasmid.... you are waiting at least for 48hours post infection of virus particle to start the treatment with puromycin..... The killing with puromycin treatment takes 72 hours after treatment and chances of any co-resistance is possible but least likely... you never know.... hope these suggestions are helpful....
Hi Aysun,
So you gradually increased the drug selection to make a Taxol resistant cells just by prolonged and gradual treatment (my guess it took you couple of month...!!!!). I am assuming you haven't already "knocked out" using a CRISPR/Cas9 system if so then you are planing to use a CRISPR/Cas9 system to knock out some gene, where you have puro selection gene in CRISPR/Cas9 vector as a positive clone selection. On the other hand, if you have already knocked out the gene then you must have used the puro selection to make those cells and then no matter how much you increase the concentration cells will NOT die....... Assuming I am right so far then the question is "Have you checked your Taxol resistant zombies could be killed by any drug at all....?" If so then try to find a CRISPR/Cas9 vector with that specific drug resistance gene.... and if you are still not able to kill those Zombies or Frankenstein you have created then send your name to Nobel nomination committee.... reason, that you have found the evidence of a prolonged drug treatment of cancer patients are NOT helping, but making an indestructible zombies....!!!!
Hi Aysun!
I am wondering whether you have found a solution or not. I have a similar problem right now, untransduced cells are somehow resitant to puromycin selection while in nontreated plate all cells are dead. As other people form my lab also use the same puromycin stock, puromycin should be fine. Any suggestions would help.
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