Expression of heme oxygenase-1 and GFP gene mediated by recombinant adeno-associated-virus in transplanted liver in rats / 中华外科杂志
Chinese Journal of Surgery
; (12): 851-853, 2008.
Article
in Zh
| WPRIM
| ID: wpr-258400
Responsible library:
WPRO
ABSTRACT
<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>
Full text:
1
Database:
WPRIM
Main subject:
Plasmids
/
Recombination, Genetic
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Transfection
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Liver Transplantation
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Rats, Wistar
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Dependovirus
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Green Fluorescent Proteins
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Heme Oxygenase-1
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Genetic Vectors
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Genetics
Limits:
Animals
Language:
Zh
Journal:
Chinese Journal of Surgery
Year:
2008
Document type:
Article