Expression of human eNOS in cardiac and endothelial cells.
In this chapter we provide a detailed technique-focused protocol for expression of human endothelial nitric oxide synthase (eNOS) gene delivered by replication-deficient adenovirus. It includes construction of recombinant plasmid with human eNOS gene full-length cDNA (heNOS), packaging of adenovirus with heNOS (Adv-heNOS) in 293 cells, confirmation of Adv-heNOS, amplification of high-titer stockAdv-heNOS, determination of the Adv-heNOS titer, expression of Adv-heNOS in the human abdominal aorta endothelial (HAAE1) cell line and rat heart myoblast cell line (H9C2), and measurement of human eNOS activity and NO production.
Wang
Xiuqing
X
Department of Medicine, University of Florida, Gainesville, FL, USA.
Cade
Robert
R
Sun
Zhongjie
Z
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001012:Aorta, Abdominal / Q000166:cytology; D015153:Blotting, Western; D002460:Cell Line; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D032446:Myoblasts / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D009206:Myocardium / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052250:Nitric Oxide Synthase Type III; D010957:Plasmids; D016133:Polymerase Chain Reaction; D051381:Rats
C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat