Abstract
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 stock Adv-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.
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References
Palmer, R. M., Ashton, D. S., and Moncada, S. (1988) Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature 333, 664–666.
Leone, A. M., Palmer, R. M., Knowles, R. G., Francis, P. L., Ashton, D. S., and Moncada, S. (1991) Constitutive and inducible nitric oxide synthases incorporate molecular oxygen into both nitric oxide and citrulline. J. Biol. Chem. 266, 23,790–23,795.
Griffith, O. W. and Stuehr, D. J. (1995) Nitric oxide synthases: properties and catalytic mechanism. Annu. Rev. Physiol 57, 707–736.
Rees, D. D., Palmer, R. M., and Moncada, S. (1989) Role of endothelium-derived nitric oxide in the regulation of blood pressure. Proc. Natl. Acad. Sci. USA 86, 3375–3378.
Lin, K. F., Chao, L., and Chao, J.(1997) Prolonged reduction of high blood pressure with human nitric oxide synthase gene delivery. Hypertension 30, 307–313.
Robert, D. G. and Robert, S. M. (1995) Adenovirus vectors, in DNA Cloning 4 (Glover, D. M. and Hames, B. D., eds.), IRL Oxford University, UK.
Nabel, E. G. and Nabel, G. J. (1994) Complex models for study of gene function in cardiovascular biology. Annu. Rev. Physiol. 56, 741–761.
Schneider, M. D. and French, B. A. (1993) The advent of adenovirus: gene therapy for cardiovascular disease. Circulation 88, 1937–1942.
Smith, R. S. Jr., Lin, K. F., Agata, J., Chao, L., and Chao, J. (2002) Human endothelial nitric oxide synthase gene delivery promotes angiogenesis in a rat model of hindlimb ischemia. Arterioscler. Thrombo. Vasc. Biol. 22, 1279–1285.
Alexander, M. Y., Brosnan, M. J., Hamilton, C. A., et al. (1999) Gene transfer of endothelial nitric oxide synthase improves nitric oxide-dependent endothelial function in a hypertensive rat model. Cardiovas. Res. 43, 798–807.
Shah, V., Chen, A. F., Cao, S., et al. (2000) Gene transfer of recombinant endothelial nitric oxide synthase to liver in vivo and in vitro. Am. J. Physiol. 279, G1023–G1030.
Jones, N. and Shenk, T. (1979) Isolation of adenovirus type 5 host range deletion mutants defective for transformation of rat embryo cells. Cell 17, 683–689.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (ed.) (1989) Molecular Cloning, A Laboratory Manual, Second ed. Cold Spring Harbor, Cold Spring Harbor Laboratory Press, New York.
Madamanchi, N. R. and Runge, M. S. (2000) Western blotting, in Angiotensin Protocols (Wang, D. H., ed.), Humana, Totowa, NJ, pp. 245–256.
Sun, Z., Cade, R., Zhang, Z., Alouidor, J., and Van, H. (2003) Angiotensinogen gene knockout delays and attenuates cold-induced hypertension. Hypertension 41, 322–327.
Cernadas, M. R., de Miguel, L. S., Garcia-Duran, M. G., et al. (1998) Expression of constitutive and inducible nitric oxide synthase in the vascular wall of young and aging rats. Circ. Res. 83, 279–286.
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Wang, X., Cade, R., Sun, Z. (2005). Expression of Human eNOS in Cardiac and Endothelial Cells. In: Sun, Z. (eds) Molecular Cardiology. Methods in Molecular Medicine™, vol 112. Humana Press. https://doi.org/10.1007/978-1-59259-879-3_7
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DOI: https://doi.org/10.1007/978-1-59259-879-3_7
Publisher Name: Humana Press
Print ISBN: 978-1-58829-363-3
Online ISBN: 978-1-59259-879-3
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