I am working with mammalian cells like MEF and also with Mesenchmymal stromal cells for transfection of a particular gene which has neomycin as a selectable marker. Several reports suggest that neomycin is toxic for mammalian cell culture. What is the alternative? What should i use for selection?
Neomycin is not a good antibiotic for selection of mammalian cells. Read this paper, which ranks antibiotics for human cell lines as "Zeocin™ > hygromycin B ∼ puromycin > neomycin" http://www.ncbi.nlm.nih.gov/pubmed/23450727
Thank you for the detail, in the paper they say zeocin is better than neomcyin now my question is if the gene of interest has neomycin as a select-able marker can i use zeocyin??
Please read this paper, hope it gives you an idea.
http://dare.uva.nl/document/228952
The Neomycin resistance gene works fine and has been used in thousands of papers and with ES cells for homologous recombination. You just need to titrate G418 concentrations which range anywhere from 100 ug/ml to 1000 ug/ml of media. Pick the lowest dose that gives you maximum killing. Cells should die within 3-5 days and colonies appear in about 10-14 days depending on how quickly your cells divide.
Yes you can, by adding G418 sulfate to the culture medium after titrating as Gregory says. However if you can choose the selection marker, I would suggest puromycin since it gives you a much quicker selection. Here're some useful tips to start with : http://www.puromycin.com/protocols.htm . If you are working with adherent cells, puromycin is definitively the best.
G418 is often used to select stable transfectant,however,in my experience, it do not guarantee to get a bona fide stable transfectant when all the G418 sensitive cells are eradicated.I transfected MCF7 with a plasmid bear neo and EGFP-tagged target protein,and use G418 in a final concentration of 750ug/ml.A week after selection,when all the control cells(untransfected) die,there is a significant portion of survivals do not bear EGFP signals.These cells can also grow quite well when colonized into a single well with a high G418 concentration.So I think G418 can only served as primary selection.
Thank you all for your valuable suggestions. i appreciate your help.
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