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.
Fujimori
Kazuhiro E
KE
Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Nakoji 3-11-46, Amagasaki, Hyogo 661-0974, Japan. k-fujimori@aist.go.jp
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D003001:Cloning, Molecular; D030541:Databases, Genetic; D004296:Dopa Decarboxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000378:metabolism; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009990:Oryzias / Q000196:embryology / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D010802:Phylogeny; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment; D015027:Zebrafish
D029941:Fish Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004296:Dopa Decarboxylase