Characterization of the regulatory region of the dopa decarboxylase gene in Medaka: an in vivo green fluorescent protein reporter assay combined with a simple TA-cloning method.

The mechanism by which differentiated cells cooperatively express specific sets of genes in multicellular organisms is a fundamental question for biologists. Currently, the mechanism is primarily attributed to complex regulation of transcriptional machinery. Here, I provide a method for studying spatiotemporal characteristics of promoters in vivo by rapid construction of reporter gene-expression vectors based on simple TA-cloning using an in vivo eGFP reporter assay in Medaka (Oryzias latipes). As an application of this method, I focused on the dopa decarboxylase (Ddc) gene, an essential enzyme for production of neurotransmitters, dopamine, and serotonin. Based on the known structure of the Medaka genome, I predicted and cloned the approximately 3 kbp fragment flanking the Ddc gene. Using an eGFP reporter assay in vivo, I showed that it functions as a promoter, directing reporter gene expression in the brain, retina, epiphysis, and gut, but not in sympathetic ganglia, kidney, or liver. Thus, the procedure presented here provides a useful tool for rapid screening of possible promoter regions and for establishing germ line-transmitted transgenic lines of Medaka.