I am working on the transfection of a murine cell line. I have to transfect 4 individual genes, so I have 4 individual plasmids, each with a different antibiotic resistance gene.
Before proceeding with the transfections I establish the optimal antibiotic concentration to kill all untransfected cells within a week, maximum two. I do this by titrating the antibiotics with my target cells; I seed them in 6 well plates and, the following day, I start treating them with the recommended antibiotic concentration ranges.
I had no problem determining the optimal concentrations for G418 (500ug/mL) and hygromycin B (300ug/mL). However, I am struggling with puromycin and blasticidin. I have spent months repeating the titrations even with higher concentrations than those recommended.
For puromycin, the recommended working concentrations are around 0.5-10ug/mL; for blasticidin, these can range between 1-30ug/mL. My cells seem to die with puromycin at a concentration of 20ug/mL, and even with this concentration they take over 2 weeks to die. For blasticidin, I am currently trying concentrations of 30, 40, 50 and 60ug/mL and, after 1 week, the cells bounce back to around 80% confluence (they died slightly but quickly recovered).
Do you have any advice? Do i try higher concentrations than these? I just don't know what could be going wrong. I replace the selection medium three times a week (Monday, Wednesday and Friday). Antibiotics are all in date. Any suggestions are welcome! Thank you.
Hello Carles Puig Saenz
Yes, it is possible. The differences in toxicity might be caused by differences in the cellular uptake and binding of the drug. There could be species-related differences in cellular toxicity possibly due to mutants exhibiting a multi-drug resistance phenotype showing increased resistance.
You may want to refer to the article attached below for more information.
https://core.ac.uk/reader/81135780
Best.
You might want to check if your murine cell line is already expressing an antibiotic resistance gene that you're not aware of. For example, 293T cells express the Neo resistance gene which means they are resistant to G418/geneticin.
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