Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Establishment of the expression system of human HtrA2 in the zebrafish

Zebrafish 동물모델에서 human HtrA2의 expression system 정립에 관한 연구

  • 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)
  • 조성원 (가톨릭대학교 생명의과학과,가톨릭대학교 분자유전학연구소) ;
  • 박효진 (가톨릭대학교 분자유전학연구소,고려대학교 생명과학대학 생명과학부) ;
  • 김구영 (가톨릭대학교 생명의과학과,가톨릭대학교 분자유전학연구소) ;
  • 남민경 (가톨릭대학교 생명의과학과,가톨릭대학교 분자유전학연구소) ;
  • 김호영 (가톨릭대학교 생명의과학과,가톨릭대학교 분자유전학연구소) ;
  • 고인호 (가톨릭대학교 분자유전학연구소) ;
  • 김철희 (충남대학교 생명과학부 생물학과) ;
  • 임향숙 (가톨릭대학교 생명의과학과,가톨릭대학교 분자유전학연구소)
  • Published : 2006.07.31
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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.

Mitochondrial serine protease로 알려진 human HtrA2 (hHtrA2)는 apoptosis 유도 과정에서 중요한 역할을 담당하고 있을 뿐만 아니라 hHtrA2가 motor neuron degeneration과 관련이 있다는 최근 연구 결과가 있으나, hHtrA2의 생리적 기능은 아직 명확하게 밝혀져 있지 않다. 이와 같이 생체내에서 필수적인 업무를 담당하는 hHtrA2의 기능을 심도 있게 연구하기 위해서는 적절한 동물모델 시스템이 필요하나 이에 대한 연구도 미흡한 실정이다. 따라서 본 연구에서는 hHtrA2의 기능 분석을 위한 기본적인 실험으로 zebrafish라는 동물모델을 선택하여 hHtrA2의 발현 시스템을 정립하였다. 먼저 zebrafish에 hHtrA2를 발현시키기 위하여 zebrafish에서 일반적으로 사용되는 발현 시스템인 pCS2+ vector에 hHtrA2와 GFP를 cloning하고 plasmid를 HEK293 cell에 transfection한 후, hHtrA2-GFP fusion 단백질의 발현을 immunoblot과 immunofluorescence staining assay로 확인한 바 약 64 kDa의 hHtrA2 단백질의 발현을 확인할 수 있었다. Zebrafish에서 hHtrA2-GFP fusion 단백질의 발현양상은 immunofluorescence microscope으로 확인하였다. hHtrA2-GFP DNA와 mRNA를 zebrafish embyro에 microinjection하여 두 가지 component의 발현을 비교 분석한 결과, DNA는 dot 형태로 mRNA는 몸 전체에 퍼져보이는 형태로 발현 양상의 차이는 있었으나 둘 다 zebrafish embryo에서 잘 발현되는 것을 알 수 있다. 다음 DNA를 주 component로 microinjection하여 zebrafish embryo에서 발현을 확인한 결과 hHtrA2는 72 hpf 까지 발현이 지속되는 것을 확인하였다. 본 연구에서 정립한 hHtrA2의 zebrafish 발현 조건은 앞으로 zebrafish에서 hHtrA2의 생리적 기능을 심도있고 정확하게 연구하는 데 있어 기본적인 자료로 활용 할 수 있을 것이다.

Keywords

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