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http://dx.doi.org/10.5352/JLS.2006.16.4.571

Establishment of the expression system of human HtrA2 in the zebrafish  

Cho, Sung-Won (Department of Biomedical Sciences, the Catholic University of Korea,Research Institute of Molecular Genetics, College of Medicine, the Catholic University of Korea)
Park, Hyo-Jin (Research Institute of Molecular Genetics, College of Medicine, the Catholic University of Korea,School of Life Science and Biotechnology, Korea University)
Kim, Goo-Young (Department of Biomedical Sciences, the Catholic University of Korea,Research Institute of Molecular Genetics, College of Medicine, the Catholic University of Korea)
Nam, Min-Kyung (Department of Biomedical Sciences, the Catholic University of Korea,Research Institute of Molecular Genetics, College of Medicine, the Catholic University of Korea)
Kim, Ho-Young (Department of Biomedical Sciences, the Catholic University of Korea,Research Institute of Molecular Genetics, College of Medicine, the Catholic University of Korea)
Ko, In-Ho (Research Institute of Molecular Genetics, College of Medicine, the Catholic University of Korea)
Kim, Cheol-Hee (Department of Biology, Chungnam National University)
Rhim, Hyang-Shuk (Department of Biomedical Sciences, the Catholic University of Korea,Research Institute of Molecular Genetics, College of Medicine, the Catholic University of Korea)
Publication Information
Journal of Life Science / v.16, no.4, 2006 , pp. 571-578 More about this Journal
Abstract
HtrA2/Omi, a mitochondrial trypsin-like serine protease, is pivotal in regulating apoptotic cell death. Several lines of recent evidence suggest that HtrA2 is associated with the pathogenesis of neurodegenerative disorders; however, the physiological function of HtrA2 still remains elusive. For studying physiological function of HtrA2 in depth, it is necessary to develop a suitable expression system in the model animal. We therefore utilized the zebrafish as a model animal to establish expression of human HtrA2 (hHtrA2) in vivo. For expression of mature HtrA2 as GFP fusion in zebrafish embryos, the HtrA2 (WT) or (S306A) cDNAs with the C-terminal GFP tag were inserted into the pCS2+ plasmid. Expression patterns of HtrA2 in HEK293 cells were first monitored by immunofluorescence staining and immunoblot assays, showing approximately 64 kDa of the HtrA2-GFP fusion proteins. Subsequently, the hHtrA2 plasmid DNA or in vitro transcribed mRNA was microinjected into zebrafish embryos. The expression patterns of HtrA2 in Zebrafish embryos were monitored by GFP fluorescence in 24 hours-post-fertilization (hpf). Although expression patterns of HtrA2-GFP in developing embryos were different between the injected DNA and mRNA, both nucleic acids revealed good expression levels to further study the physiological role of HtrA2 in vivo. This study provides a suitable condition for expressing hHtrA2 in the zebrafish embryos as well as a method for generating useful system to investigate physiological properties of the specific human genes.
Keywords
Human HtrA2; zebrafish; embryos; expression system; serine protease;
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