Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Expression of Human Cytochrome b5 in Zebrafish

Zebrafish에서 human cytochrome b5의 발현

  • Han, Se Mi (Department of Biology, Keimyung University) ;
  • Yoo, Min (Department of Biology, Keimyung University)
  • 한세미 (계명대학교 생물학과) ;
  • 유민 (계명대학교 생물학과)
  • Received : 2017.01.19
  • Accepted : 2017.02.15
  • Published : 2017.06.30
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Abstract

In this study, we sought to develop an effective cloning system by which human cytochrome $b_5$ (cyt $b_5$) is introduced and expressed in zebrafish. First, the 414 bp human cyt $b_5$ gene was amplified from RNA extracts of HeLa cells using RT-PCR, and the amplicon was subsequently sequenced to confirm that it was intact. Next, cyt $b_5$ was cloned into the pEGFP-N3 vector, which also encodes a fluorescent gene. One-cell stage zebrafish embryos were microinjected with the recombinant vector containing the cyt $b_5$ gene. Fluorescence microscopy confirmed high expression of the fluorescent gene in the injected fry compared to the non-fluorescent control fry. Finally, we extracted RNA from the injected fry and performed RT-PCR to determine whether the human cyt $b_5$ gene is expressed in the transgenic zebrafish. Sequencing analysis further confirmed that the cloned human cyt $b_5$ gene was intact. The transgenic zebrafish model produced in this study will be a useful tool to study therapeutic approaches to cure various diseases related to the deficiency of functional human cyt $b_5$ as well as tools for cloning useful genes in fish.

본 연구에서는 zebrafish에 사람의 cyt $b_5$ 유전자를 microinjection하여 발현시키고, 그 결과를 형광으로 확인하였다. HeLa cell에서 RT-PCR을 진행한 결과 414 bp의 cyt $b_5$ band가 증폭되었다. 염기서열 분석으로 재확인된 cyt $b_5$ insert를 pEGFP-N3의 형광 vector에 클로닝하였고, 이렇게 준비된 pEGFP-N3-cyt $b_5$ plasmid DNA를 1세 포기의 수정란에 microinjection하였다. cyt $b_5$를 microinjection한 치어를 형광현미경으로 관찰한 결과 대조군 치어보다 훨씬 선명한 형광을 띠는 것이 확인되었다. 최종적으로 치어에서 RNA를 분리하여 RT-PCR하였고 전기영동과 DNA sequencing으로 fusion 단백질의 발현을 재확인하였다. Cyt $b_5$의 발현으로 인해 zebrafish의 생존율이 다소 떨어지는 것으로 확인되었기에 독성 문제를 해결하기 위한 연구가 계속 필요할 것으로 사료된다. 이 연구는 향후 cyt $b_5$가 결핍되었을 경우 발생할 수 있는 여러 질병들을 유전자 차원에서 치료하고, 유용 유전자 클로닝을 위한 기술 개발에 발판이 될 수 있을 것으로 기대된다.

Keywords

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