Optimisation of gene transfer into vascular endothelial cells using electroporation

Abstract

Objectives: We have examined the conditions required to obtain optimum transfection efficiencies for human umbilical vein endothelial cells by transduction with a plasmid conferring neomycin resistance.

Materials and methods: Preliminary studies examined the effects of electric discharges using the Biorad Gene Pulser on endothelial cells. Post-electroporation, there was a significant decrease in cell survival with increasing voltages (100-400 volts; p = 0.03), capacitances [125-960 microFarads (microF); p = 0.02], number of electric pulses (1-2; p = 0.03) and decreasing cell concentrations (p = 0.01). The optimal cell concentration was 3 x 10(6) cells/ml. Transfection studies utilised the neomycin resistance expressing plasmid, pTCF; transfectants were selected with the neomycin analogue G-148.

Results: Electro-transfection was optimised with increasing voltages (p = 0.02) and capacitances (p = 0.01) using a single pulse. Optimal transfection was obtained using 400 volts with a capacitance of 960 microF using a single pulse; the median transfection efficiency was 10%. Transduced endothelial cells stably expressed the plasmid for 12 days and at least two cell passages.

Conclusions: The results indicate that endothelial cells can be efficiently transduced by electroporation to stably express an introduced gene. This may have important implications in vascular surgery.