Abstract
The transfer and expression of DNA plasmids containing promoter fragments of heterologous genes linked to reporter cDNAs in mammalian cells has become an invaluable technique for studying the regulation of gene expression. Several reporter genes such as luciferase, β-galactosidase, chloramphenicol acetyl transferase, and green flourescent protein are ideal to study promoter activities as their gene products are not endogenous to smooth muscle cells (SMC) and their expression can be readily detected using convenient assays (1). Among these genes, a popular choice is the firefly luciferase, as its expression can be easily detected in cells using a highly sensitive chemiluminescent assay (2). The firefly luciferase catalyses a rapid, ATP-dependent oxidation of the substrate, luciferin, which then emits light. Reactions catalyzed by firefly luciferase are:
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ausubel, F., Brent, R., Kingston, R., Moore, D., Seidman, J., Smith, J., and Struhl, K., ed. (1994), Current Protocols in Molecular Biology. Wiley, USA.
Gould, S. and Subramani, S. (1988) Firefly luciferase as a tool in molecular and cell biology. Anal. Biochem. 175, 5–13.
De Wet, J. R., Wood, K. V., DeLuca, M., Helinski, D. R., and Subramani, S. (1987) Firefly luciferase gene: Structure and expression in mammalian cells. Mol. Cell. Biol. 7, 725–737.
Alam, J. and Cook, J. (1990) Reporter genes: application to the study of mammalian gene transcription. Anal. Biochem. 188, 245–254.
MacGregor, G., GP, N., Fiering, S., Roederer, M., and Herzenberg, L. (1991) Use of E. coli lacZ (β-galactosidase) as a reporter gene, in Gene Transfer and Expression Protocols (Murray, E., ed.), Humana Press, Totowa, NJ, pp. 217–235.
Jain, V. and Magrath, I. (1991) A chemiluminescent assay for quantitation of beta-galactosidase in the femtogram range: application to quantitation of beta-galactosidase in lacZ-transfected cells. Anal. Biochem. 199, 119–124.
Young, D., Kingsley, S., Ryan, K., and Dutko, F. (1993) Selective inactivation of eukaryotic beta-galactosidase in assays for inhibitors of HIV-1 TAT using bacterial beta-galactosidase as a reporter enzyme. Anal. Biochem. 215, 24–30.
Felgner, P. L. and Ringold, G. M. (1989) Cationic liposome-mediated transfection. Nature 337, 387–388.
Hawley-Nelson, P., Ciccarone, V., Gebeyehu, G., and Jessee, J. (1993) Lipofectamine reagent: A new, higher efficiency polycationic liposome transfection reagent. Focus 15, 73–79.
McMurray, H., Parrott, D. P., and Bowyer, D. E. (1991) A standardised method of culturing aortic explants, suitable for the study of factors affecting the phenotypic modulation, migration and proliferation of aortic smooth muscle cells. Atherosclerosis 86, 227–237.
Southgate, K. and Newby, A. C. (1990) Serum-induced proliferation of rabbit aortic smooth muscle cells from the contractile state is inhibited by 8-Br-cAMP but not 8-Br-cGMP. Atherosclerosis 82, 113–123.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1999 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Fabunmi, R.P. (1999). Use of Liposome-Mediated DNA Transfection to Determine Promoter Activity in Smooth Muscle Cells. In: Baker, A.H. (eds) Vascular Disease. Methods in Molecular Medicine™, vol 30. Humana Press. https://doi.org/10.1385/1-59259-247-3:143
Download citation
DOI: https://doi.org/10.1385/1-59259-247-3:143
Publisher Name: Humana Press
Print ISBN: 978-0-89603-731-1
Online ISBN: 978-1-59259-247-0
eBook Packages: Springer Protocols