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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

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Abstract

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.

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Abbreviations

Ddc :

Dopa decarboxylase

Hdc :

Histamine decarboxylase

eGFP :

Enhanced green fluorescent protein

MGD:

Medaka genome database

Dpi:

Day(s) post injection

Dpf:

Day(s) post fertilization

UTR:

Untranslated region

Bp:

Base pairs

CNS:

Central nervous system

PNS:

Peripheral nervous system

β-lac:

Beta-lactamase

PCR:

Polymerase chain reaction

HB:

Hybridization buffer

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Acknowledgment

I would like to thank Yuji Ishikawa for providing Hd-rR and Yuko Wakamatsu for providing SeeThrough via the National BioResource Project (http://www.nbrp.jp/). I also thank Yoriko Jyosaki, Takako Iwanaga, and Kaoru Ogata for excellent assistance in maintaining the fish. This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (#18650095, K.E.F).

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  1. Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Nakoji 3-11-46, Amagasaki, Hyogo, 661-0974, Japan

    Kazuhiro E. Fujimori

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Correspondence to Kazuhiro E. Fujimori.

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Fujimori, K.E. 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. Mol Biotechnol 41, 224–235 (2009). https://doi.org/10.1007/s12033-008-9120-1

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