I have tried to read about this but I don't find definitive answers.
I am trying to lentivirally transduce a mouse cell line with a few different plasmids (each encoding a different antibiotic resistance gene, namely puromycin, hygromycin and G418). After successfully transducing the cells a first time (with the puromycin resistance-containing plasmid), it now looks like when I transduce the cells with the hygromycin resistance-containing plasmid they don't die at the hygromycin concentration established during the antibiotic titration.
Has anyone experienced this?
I found this paper, but it doesn't seem to answer my question fully: https://www.sciencedirect.com/science/article/abs/pii/S0003269709008434
Hi Carles Puig,
I haven't faced a problem like that. However I can share you some considerations:
1) Have you considered non-simultaneos transduction of your plasmids? For instance, first you transduce a plasmid, then you perform a selection of 2-3 days, then another plasmid with its subsequent selection and so on...
2) In our lab we had a couple of problems with G418, it was because of the analogy with neomycine and hygromicine. Maybe you can check thoroughly if the gene resistance can show cross-reaction, and if so, change one of the antibiotics
What type of cells are you transducing/transfecting?
I hope it helps
Best regards,
Francesc
Thanks for your answer Francesc.
I am indeed trying to transduce the cells one plasmid at a time. However my selection periods are longer (a few weeks, until my non-transduced control cells die). Then I assess expression by western blot and, if positive, I begin the next transduction.
I am transducing a mouse cancer cell line (Py8119), and I am meant to transduce it several times (4 plasmids, each with a different resistance gene: neomycin, hygromycin, puromycin and zeocin).
My main concern is that the initial transduction (which was successful) was done with the plasmid containing the puromycin resistance gene and, annoyingly, these cells are naturally quite resistant to puromycin (some literature uses 10ug/mL, but according to my own titration they only die with 20ug/mL, obviously a very high concentration). I wonder whether this is causing the cells to express drug efflux pumps or some resistance mechanism…
I am currently trying to transduce the original cells with the plasmid containing the neomycin resistance gene and see how it goes. The amount of G418 required is 500ug/mL according to my titration.
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