Construction of a recombinant vector based on AAV carrying human endothelial nitric-oxide synthase gene.
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.
He
Chen-Xia
CX
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, China.
Miao
Chao-Yu
CY
Yao
Ji-Hua
JH
Chen
Hao-Ming
HM
Lu
Da-Ru
DR
Su
Ding-Feng
DF
Xue
Jing-Lun
JL
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
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