Expression of heme oxygenase-1 and GFP gene mediated by recombinant adeno-associated-virus in transplanted liver in rats.
- Author:
Liang SUN
1
;
Hai-quan QIAO
;
Tie-feng SHI
;
Xian JIANG
;
Bo TANG
;
Hong-chi JIANG
;
Xue-ying SUN
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Dependovirus; genetics; Female; Genetic Vectors; Green Fluorescent Proteins; genetics; metabolism; Heme Oxygenase-1; genetics; metabolism; Liver; metabolism; Liver Transplantation; Male; Plasmids; genetics; Rats; Rats, Wistar; Recombination, Genetic; Transfection
- From: Chinese Journal of Surgery 2008;46(11):851-853
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo 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.
METHODSHeme 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.
RESULTSThe 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.
CONCLUSIONSHigh 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.