Dual luciferase reporter system

FIGURE 20.2 The involvement of RARE on apo-lO -lycopenoic acid-transactivated RARp expression. Upper panel Diagram of the RARp reporter vector with wild type and mutated R AREs. Lower panel HeLa cells transfected with the RARp reporter vector and an internal control vector were treated with 5 pmol/I. of apo-lO -lycopenoic acid or 1 pmol/L of all-trans retinoic acid for 24h. Luciferase activities were measured by dual-luciferase reporter system. Values are means of SEM of three replicate assays., statistically significantly different, as compared with control in the same group, P < 0.05. (Adapted from Lian, F. et al., Carcinogenesis, 28, 1567, 2007. With permission.)... 

Dual-luciferase reporter system (Promega, Madison, WI). 

Cells are lysed for Firefly and Renilla luciferase assays using the Dual-Luciferase Reporter Assay system (Promega), following the manufacturer s instructions. We use a multimode microplate reader with automatic injectors (FLUOROstar Optima from BMG Labtech, OfFenburg, Germany) for luminescence measurements. 

Williams, T.D., J.S. Lee, D.L. Sheader and J.K. Chipman. The cytochrome P450 1A gene (CYP1A) from European flounder (Platichthys flesus), analysis of regulatory regions and development of a dual luciferase reporter gene system. Mar. Environ. Res. 50 1—6, 2000. 

Dyer, B. W., Ferrer, F. A., Klinedinst, D. K., and Rodriguez, R. (2000). A noncommercial dual luciferase enzyme assay system for reporter gene analysis. Anal. Biochem. 282, 158-161. 

The distinct difference in substrate makes it possible to use both luciferase reporters simultaneously in one assay, as in Promega s Dual-Ludferase Reporter (DLR ) assay system [162]. 

This chapter covers j8-galactosidase (jSGal or LacZ) and luciferase (Lux/Luc) gene systems as typical reporter systems. Note that alkaline phosphatase (PhoA) can also be used as an excellent reporter system and has been described in Chapter 5. As selection markers, the ampicillin resistance system (Bla or ApO will be described. The chloramphenicol acetyltransferase (CAT) gene system will also be discussed as a typical marker/reporter with dual functions. 

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