Optimisation of gene transfer into vascular endothelial cells using electroporation.
OBJECTIVE
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.


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.
Kotnis
R A
RA
Department of Surgery, University of Leicester, Leicester Royal Infirmary, U.K.
Thompson
M M
MM
Eady
S L
SL
Budd
J S
JS
Bell
P R
PR
James
R F
RF
D002470:Cell Survival; D018274:Electroporation; D004730:Endothelium, Vascular / Q000166:cytology*; D018014:Gene Transfer Techniques; D006801:Humans; D009355:Neomycin; D011815:R Factors / Q000235:genetics; D014162:Transfection / Q000379:methods; D014471:Umbilical Veins / Q000166:cytology*
D009355:Neomycin