Dual-reporter assay using two secreted luciferase genes.

Luciferase is used as a reporter enzyme to estimate gene expression in prokaryotic or eukaryotic cells because the amount of luciferase present is correlated with light intensity in the presence of excess luciferin (1). Furthermore, its sensitivity and range of linear responses are superior to those of other typical reporters (2). When the luciferase gene containing a target promoter region is transfected into target cells using a plasmid, expression of luciferase can be estimated from bioluminescence after 1 or 2 days. For practical use, two or three promoters are used for the target and control genes because the expression of a reporter enzyme depends on conditions in the cell, which can be estimated from expression of a control gene. The dual luciferase reporter assay system (www.promega.com/tbs/tm040/tw040. html) involves the use of firefly luciferase for monitoring gene expression and another type of luciferase [e.g., sea pansy (Renilla) luciferase] as an internal control. Recently, Nakajima et al. developed a tricolor-reporter in vitro assay system in which expression of three genes is monitored simultaneously by splitting emissions from green-, orange-, and red-emitting beetle luciferases using optical filters (3). However, neither in vitro assay system is suitable for high-throughput analysis of multiple gene expression, because they involve sample lysis or require special equipment, such as a filtered luminometer. In this report, we describe a new dual-reporter assay involving two secreted luciferases that does not require lysis or special equipment. The marine ostracod crustacean Cypridina and the marine copepod Gaussia secrete light-blue luminescent luciferases into seawater (4,5). When cDNA for Cypridina luciferase (CLuc; λmax = 460 nm) is transfected into mammalian cells, the CLuc is secreted into the medium via the endoplasmic reticulum (ER) and the Golgi complex (6). Gaussia luciferase (GLuc; λmax = Dual-reporter assay using two secreted luciferase genes