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Electroporation of Primary Bone Marrow Cells

nomic DNA.

We have also used the Gene Pulser apparatus to stably transfect adherent 929 cells with the dhfr gene in order to compare the conditions required when an adherent cell rather than a nonadherent one was the target. These cells were cultured in medium containing MTX to determine the percentage of cells expressing the mutant enzyme. The use of the phosphate buffered sucrose (272 mM sucrose, 7 mM PO4, 1 mM MgCl2, pH 7.4) allowed a lower capacitance setting of 25 F; and with a 250 V (650 V/cm) pulse, a time constant of 8 -11 msec was optimal. These Gene Pulser settings resulted in the production of the mutant dhfr in 19% of the cells capable of forming colonies, a 4-fold increase over controls. Table 2 gives a comparison of the parameters we have found to give optimal results for both adherent and non-adherent cell types.

In our system, the most important variables appear to be field strength (v/cm) and pulse time constant. We found that electroporation conditions of 625-750 V/cm and time constants of 7-11 msec were optimal for transient expression or stable transfection of bone marrow or L929 cells. With phosphate buffered sucrose electroporation medium, the 25 F capacitor was used to produce a pulse with a time constant in the optimal range. When lower resistance PBS was used, the 250 and 500 F capacitors of the Capacitance Extender were required. The conditions reported here are now used routinely in our laboratory in our studies of the regulation of class II gene expression and the immune response.


References
1. The mutant dhfr sequence, pFR400, was generously provided by Genentech, Inc.,
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