Increased efficiency of transfection of murine hybridoma cells with DNA by electropermeabilization.
Dispase-treated murine hybridoma cells (SP2/0-Ag14) were transfected with the G418 resistance gene bearing plasmid pSV2-neo by electropermeabilization with a high degree of efficiency. The cells were subjected to intermittent multiple high-voltage short duration (5 microsecond) DC pulses at intervals of 1 min in a weakly conducting medium followed by selection in G418-containing medium. The transfection medium, temperature, pulse duration, and voltage were empirically determined by preliminary electropermeabilization experiments. Increasing the number of pulses resulted in a higher percentage of transfected cells, but a decrease in the number of viable cells, with the optimal transfectant yield resulting when five pulses of 10 kV/cm were administered. This method allows the rapid and efficient injection of DNA into mammalian cells, and permits the rapid production of stable, drug resistant hybridoma cell lines for use in subsequent fusion experiments.
Stopper
H
H
Lehrstuhl für Biotechnologie, Universität Würzburg, F.R.G.
Zimmermann
U
U
Neil
G A
GA
D000818:Animals; D002463:Cell Membrane Permeability; D002470:Cell Survival; D004274:DNA, Recombinant / Q000378:metabolism*; D004560:Electricity; D006825:Hybridomas / Q000378:metabolism* / Q000502:physiology; D051379:Mice; D008845:Microinjections; D011815:R Factors; D014162:Transfection
D004274:DNA, Recombinant