Use of Firefly Luciferase Reporter Gene to Study Angiotensinogen Acute Phase Response Element

Abstract

This chapter describes the use of firefly luciferase reporter gene to study angiotensinogen acute phase response element. Analysis of regulatory sequences of DNA has been greatly facilitated with the introduction of reporter genes, namely, genes that encode an easily assayble product that is not normally present in the cell lines under investigation. Firefly luciferase from the firefly lantern produces light by the ATP-dependent oxidation of luciferin. Modification of the plasmid vector, assay conditions, and incorporation of a contrasfected internal control reporter alkaline phosphatase to control for plate–to–plate transfection efficiency allows for sensitive, reproducible determinations of enhancer/promoter activity in living cells. Luciferase reporter plasmids are introduced into cultured cells using conventional techniques with the optimal method of transfection being determined empirically for individual cell lines. The transiently transfected hepatoma cells after 48 h for the reporter assay, a time at which transfected luciferase reporter activity is maximal, are harvested. Luciferase, CAT, and alkaline phosphatase reporter activity accumulate primarily in the cytoplasm of transfected cells. The firefly luciferase reporter assay provides rapid results of cellular transfections. The cytokine-inducible enhancer sequence, acute phase response element, of the rat angiotensinogen gene is identified in native sequences by site-directed mutagenesis between nucleotides.