title: Construction of a recombinant vector based on AAV carrying human endothelial nitric-oxide synthase gene.
abstract: OBJECTIVE
  To construct an AAV based vector carrying human endothelial nitric-oxide synthase (eNOS) cDNA and study its expression in vitro for future gene therapy.
  
  
  METHODS
  eNOS cDNA was inserted into the EcoR I site of pSNAV-1 containing the cytomegalovirus (CMV) promoter and inverted terminal repeat sequences of adeno-associated virus. The constructed vector was transfected into BHK and C2C12 cells. eNOS cDNA and mRNA were detected by polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR), respectively.
  
  
  RESULTS
  By restriction enzyme digestion analysis, it was proved that eNOS cDNA was inserted into pSNAV-1 in a proper direction. PCR detection demonstrated that pSNAV-eNOS was transferred into both BHK and C2C12 cells. RT-PCR analysis showed that these pSNAV-eNOS transfected cells could express eNOS mRNA.
  
  
  CONCLUSIONS
  pSNAV-eNOS was successfully constructed with the ability to express human eNOS mRNA in cultured mammalian cells.
authors.affiliation: State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, China.
authors.forename: Chen-Xia
authors.initials: CX
authors.lastname: He
authors.forename: Chao-Yu
authors.initials: CY
authors.lastname: Miao
authors.forename: Ji-Hua
authors.initials: JH
authors.lastname: Yao
authors.forename: Hao-Ming
authors.initials: HM
authors.lastname: Chen
authors.forename: Da-Ru
authors.initials: DR
authors.lastname: Lu
authors.forename: Ding-Feng
authors.initials: DF
authors.lastname: Su
authors.forename: Jing-Lun
authors.initials: JL
authors.lastname: Xue
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D000229:Dependovirus / Q000235:genetics*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000166:cytology; D051379:Mice; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D019076:Transgenes
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse