Control by pulse parameters of DNA electrotransfer into solid tumors in mice.
Electrotransfer (electroporation) is recognized as one of the most promising alternatives to viral vectors for transfection of different tissues in vivo for therapeutic purposes. We evaluated the transfection efficiency of reporter genes (green fluorescent protein and luciferase) in murine subcutaneous tumors using different combinations of high-field (HV) (600-1400 V cm(-1), 100 mus, 8 pulses) and low-field (LV) (80-160 V cm(-1), 50-400 ms, 1-8 pulses) pulses and compared it to protocol using eight identical pulses of 600 V cm(-1) and 5 ms duration (electro-gene therapy, EGT). Expression of GFP was determined using a fluorescent microscope and flow cytometry and expression of luciferase by measuring its activity using a luminometer. The EGT protocol yielded the highest expression of both reporter genes. However, a careful optimization of combinations of HV and LV pulses may result in similar transfection as EGT pulses. With the combination protocol, relatively high fields of LV pulses were necessary to obtain comparable transfection to the EGT protocol. Expression of reporter genes was higher in B16 melanoma than in SA-1 fibrosarcoma. Our data support the hypothesis that both electropermeabilization and electrophoresis are involved in electrotransfer of plasmid DNA, but demonstrate that these components have to happen at the same time to obtain significant expression of the target gene in tumors.
Cemazar
M
M
Department of Experimental Oncology, Institute of Oncology, Ljubljana, Slovenia.
Golzio
M
M
Sersa
G
G
Hojman
P
P
Kranjc
S
S
Mesojednik
S
S
Rols
M-P
MP
Teissie
J
J
D000818:Animals; D004247:DNA / Q000008:administration & dosage*; D018274:Electroporation / Q000379:methods*; D005260:Female; D005354:Fibrosarcoma / Q000378:metabolism*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008545:Melanoma / Q000378:metabolism*; D051379:Mice; D008805:Mice, Inbred A; D008810:Mice, Inbred C57BL; D010957:Plasmids; D014162:Transfection; D014407:Tumor Cells, Cultured
D049452:Green Fluorescent Proteins; D004247:DNA; D008156:Luciferases