Electroporation-mediated ex vivo gene transfer into graft not requiring injection pressure in orthotopic liver transplantation.
BACKGROUND
We investigated optimum conditions for ex vivo gene transfer into liver grafts by plasmid injection via the portal vein combined with electroporation in rat liver transplantation.


METHODS
Anesthetized 9-week-old male Shionogi-Wistar rats were used as donors and recipients. After harvest of the liver graft from the donor rat, a tapered 3Fr. catheter was inserted into the portal vein of the liver graft ex vivo. After clamping the afferent vessels around the right and caudal liver lobes, pCAGGS-luciferase, which was diluted with one of several osmotic pressure solutions, or pCAGGS-green fluorescence protein (GFP) plasmid was injected into these lobes to keep the efferent vessels patent. Electrical pulses were applied to the liver graft during cold preservation in lactated Ringer's solution, University of Wisconsin solution, and histidine-tryptophan-ketoglutarate solution.


RESULTS
Transfection efficacy was estimated by measurement of luciferase activity. Luciferase activity in the liver was dependent on both the voltage and electric current of the electrical pulse, and also on the type of preservation solution and plasmid osmotic pressure. Luciferase activity was noted only in plasmid-injected lobes of the liver graft. GFP-transfected cells were identified by GFP fluorescence. GFP was observed predominantly in perivascular cells, including hepatocytes.


CONCLUSIONS
We have demonstrated successful ex vivo gene transfection into liver grafts without injection pressure by using a non-viral method.
Kobayashi
Shogo
S
Department of Surgery and Clinical Oncology, Graduate School of Medicine, Osaka University, Osaka, Japan.
Dono
Keizo
K
Takahara
Shiro
S
Isaka
Yoshitaka
Y
Imai
Enyu
E
Zhenhui
Lu
L
Nagano
Hiroaki
H
Tomoaki
Kato
K
Umeshita
Koji
K
Nakamori
Shoji
S
Sakon
Masato
M
Monden
Morito
M
D000818:Animals; D018274:Electroporation / Q000295:instrumentation / Q000379:methods*; D015870:Gene Expression; D005822:Genetic Vectors / Q000008:administration & dosage; D049452:Green Fluorescent Proteins; D007275:Injections, Intravenous; D016031:Liver Transplantation / Q000379:methods*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008297:Male; D010957:Plasmids / Q000008:administration & dosage; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D014162:Transfection / Q000379:methods*
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases