Large scale transfection of mouse L-cells by electropermeabilization

Abstract

Mouse L-cells were transfected by electropermeabilization using the selectable plasmid pSV2-neo which confers resistance to G-418 (Geneticin). The DNA concentration used was 1 microgram/ml, the field strength was 10 kV/cm, the duration of the pulse was 5 microseconds. Transfection yield was optimal at a temperature of 4 degrees C when using a time in between consecutive pulses of 1 minute compared to shorter (of the order of seconds) or longer (3 minutes) time intervals. A more detailed study of the relationship between the number of pulses applied (up to 10) and transfection yield showed it to be almost linear in this range at 4 degrees C. The yield of transfectants in response to 10 pulses was up to 1000 per 10(6) cells (using 3.3 pg DNA per cell). The influence of the growth phase of the cells on the transfection yield and/or the subpopulation of the mouse L-cell line used was shown. Furthermore the clone yield depended on the DNA per cell ratio within a very small range.