I have been trying transfections with this cell line, with hardly any success.
Bicistronic IRES vectors don't seem to work
Electroporation has been giving low efficiencies
High concentration of G418 is needed to kill them (in order to use for selection of transfectants)
Can anyone suggest vectors, antibiotics and transfection methods that have worked for this or a similar cell line?
Hi,
I would suggest retro- or lentiviral transduction. In my hands the best results gave Nolan lab protocol http://web.stanford.edu/group/nolan/_OldWebsite/protocols/pro_optimiz.html
Best,
Lena
Viral transductions should hopefully work. But as of now we are hesitant to step into viruses because of safety and handling issues. Thank you for the suggestion.
In this case I would suggest Fugene HD, which usually works as the charm. Though you would need to do titration assay to find out what ratio (plasmid/reagent) will work the best in your system. Good luck!
Mr.Alex, do you use the same cell lines? If so, what conditions and which plasmid do you use?
Hi Devi,
I use a GFP plasmid, specifically GFP cloned into pcDNA3.1, I typically resuspend 1x10^6 M12.4.1 cells in a specifically formulated buffer that we call ZAP buffer (25mM HEPES, 0.75mM Na2HPO4, 140mM KCl, 5mM NaCl, 2mM MgCl, 0.5% w/v Ficoll 400), then pulse the cells with an exponential decay protocol. I use a 4mm cuvette and aBioRad GenePulser Xcell with 350 or 375 V, 975 micro Faradays, and infinite resistance.
Thanks a lot. I'll try that out. What is the time scale of GFP expression? I usually check from 24 to 96 hours.
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