title: [Expression of heme oxygenase-1 and GFP gene mediated by recombinant adeno-associated-virus in transplanted liver in rats].
abstract: 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.
authors.affiliation: Department of General Surgery, the First Clinical Hospital Affiliated of Harbin Medical University, Harbin 150001, China. sunliang513@tom.com
authors.forename: Liang
authors.initials: L
authors.lastname: Sun
authors.forename: Hai-quan
authors.initials: HQ
authors.lastname: Qiao
authors.forename: Tie-feng
authors.initials: TF
authors.lastname: Shi
authors.forename: Xian
authors.initials: X
authors.lastname: Jiang
authors.forename: Bo
authors.initials: B
authors.lastname: Tang
authors.forename: Hong-chi
authors.initials: HC
authors.lastname: Jiang
authors.forename: Xue-ying
authors.initials: XY
authors.lastname: Sun
mesh_terms: 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
chemical_list: D049452:Green Fluorescent Proteins; D051547:Heme Oxygenase-1