[Expression of heme oxygenase-1 and GFP gene mediated by recombinant adeno-associated-virus in transplanted liver in rats].
OBJECTIVE
To construct and purify heme oxygenase-1, GFP gene mediated by recombinant adeno-associated-virus and identify expression rate of GFP in transplanted liver in rats.


METHODS
Heme oxygenase-1 gene of rat was cloned and subcloned to rAAV vector, the gene sequence was confirmed correct by restriction enzyme and DNA sequencing. The rAAV-HO-1 was then cotransfected into 293 cell line with accessory plasmid virus helper and AAV-cap-rep through CaCl2 coprecipitation. Virus particles were purified by heparin column chromatography and titre were detected by Real-time PCR. An orthotopic liver transplantation model by Wistar to Wistar was set up using Kamada's two cuff technique. Purified rAAV-GFP was injected into portal vein and incubated for 2 hours at the donor liver cold preserved stage, and then performed OLT. Recipients were killed and visceral organs were sampled at 1 and 3 months after operation respectively, frozen section (3-5 microm) were prepared and gene expression rate in different tissues was examined under fluorescence microscope.


RESULTS
The inserted segment of HO-1 was identified through restriction enzyme cutting followed with electrophoresis, the result of DNA sequencing was in accordance with which found in Genbank. The GFP expression rate was over 80% in allograft at 1 and 3 month after transfection whereas there was no GFP expression in heart, lung, spleen, kidney and small bowel.


CONCLUSIONS
High titre rAAV carried HO-1 and GFP were constructed successfully. Steady and effective expression of GFP mediated by rAAV was demonstrated in liver allograft in rats.
Sun
Liang
L
Department of General Surgery, the First Clinical Hospital Affiliated of Harbin Medical University, Harbin 150001, China. sunliang513@tom.com
Qiao
Hai-quan
HQ
Shi
Tie-feng
TF
Jiang
Xian
X
Tang
Bo
B
Jiang
Hong-chi
HC
Sun
Xue-ying
XY
D000818:Animals; D000229:Dependovirus / Q000235:genetics*; D005260:Female; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D051547:Heme Oxygenase-1 / Q000235:genetics* / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D016031:Liver Transplantation; D008297:Male; D010957:Plasmids / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D011995:Recombination, Genetic; D014162:Transfection
D049452:Green Fluorescent Proteins; D051547:Heme Oxygenase-1