생명과학회지 (Journal of Life Science)

  • 제27권6호
  • /
  • Pages.617-622
  • /
  • 2017
  • /
  • 1225-9918(pISSN)
  • /
  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
권호 표지
DOI QR코드

DOI QR Code

Zebrafish에서 human cytochrome b5의 발현

Expression of Human Cytochrome b5 in Zebrafish

  • 한세미 (계명대학교 생물학과) ;
  • 유민 (계명대학교 생물학과)
  • Han, Se Mi (Department of Biology, Keimyung University) ;
  • Yoo, Min (Department of Biology, Keimyung University)
  • 투고 : 2017.01.19
  • 심사 : 2017.02.15
  • 발행 : 2017.06.30
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

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.

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.

키워드

참고문헌

  1. Cheng, X., Chen, X., Jin, X., He, J. and Yin, Z. 2014. Generation and characterization of gsua: EGFP transgenic zebrafish for evaluating endocrine-disrupting effects. Toxicol. Appl. Pharmacol. 278, 78-84. https://doi.org/10.1016/j.taap.2014.04.009
  2. Cho, S. W., Park, H. J., Kim, G. Y., Nam, M. K., Kim, H. Y., Ko, I. H., Kim, C. H. and Rhim, H. S. 2006. Establishment of the expression system of human HtrA2 in the zebrafish. J. Life Sci. 4, 571-578.
  3. Choi, T. Y., Kim, S. M., Sohn, K. C., Kim, C. D., Lee, J. H. and Yoon, T. J. 2007. Zebrafish as an in vivo model for the study of skin cells. J. Invest. Dermatol. 14, 37-44.
  4. Dariush, N., Fisher, C. W. and Steggles, A. W. 1988. The nucleotide sequence of rabbit liver cytochrome $b_5$ mRNA. Protein Seq. Data Anal. 1, 351-353.
  5. Douglas, R. H. and Hultquist, D. E. 1978. Evidence that two forms of bovine erythrocyte cytochrome $b_5$ are identical to segments of microsomal cytochrome $b_5$. Proc. Natl. Acad. Sci. USA 75, 3118-3122. https://doi.org/10.1073/pnas.75.7.3118
  6. Giordano, S. J. and Steggles, A. W. 1991. The human liver and reticulocyte cytochrome $b_5$ mRNAs are products from a single gene. Biochem. Biophys. Res. Commun. 178, 38-44. https://doi.org/10.1016/0006-291X(91)91776-9
  7. Giordano, S. J. and Steggles, A. W. 1993. Differential expression of the mRNAs for the soluble and membranebound forms of rabbit cytochrome $b_5$. Biochim. Biophys. Acta 1172, 95-100. https://doi.org/10.1016/0167-4781(93)90274-H
  8. Giordano, S. J., Yoo, M., Ward, D. C., Bhatt, M., Overhauser, J. and Steggles, A. W. 1993. The human cytochrome $b_5$ gene and two of its pseudogenes are located on chromosomes 18q23, 14q31-32.1 and 20p11.2, respectively. Human Gen. 92, 615-618. https://doi.org/10.1007/BF00420948
  9. Greg, C., Marcela, T., Shuo, L. and Sigrid, R. 2006. Fluorescent tagged analysis of neural gene function using mosaics in zebrafish and Xenopus laevis. Brain Res. 1070, 150-159. https://doi.org/10.1016/j.brainres.2005.11.079
  10. Hegesh, E., Hegesh, J. and Kaftory, A. 1986. Congenital methemoglobinemia with a deficiency of cytochrome $b_5$. New Engl. J. Med. 314,757-761. https://doi.org/10.1056/NEJM198603203141206
  11. Jaffe, E. R. 1981. Methemoglobinemia. Clinics Haematology 10, 99-122.
  12. Jaffe, E. R. 1985. Methemoglobinemia in the differential diagnosis of cyanosis. Hosp. Pract. 20, 92-96, 101-103, 108-110.
  13. Jagow, G. and Walter, S. 1980. b-type cytochromes. Ann. Rev. Biochem. 49, 281-314. https://doi.org/10.1146/annurev.bi.49.070180.001433
  14. Kearns, E. V., Keck, P. and Somerville, C. R. 1992. Primary structure of cytochrome $b_5$ from cauliflower (brassica oleracea) deduced from peptide and cDNA sequences. Plant Physiol. 99, 1254-1257. https://doi.org/10.1104/pp.99.3.1254
  15. Kominami, S., Noriyuki, O., Reiko, M., Huang, D. Y. and Shigeki, T. 1992. The role of cytochrome $b_5$ in adrenal microsomal steroidogenesis. J. Steroid Biochem. Mol. Biol. 42, 57-64. https://doi.org/10.1016/0960-0760(92)90011-7
  16. Li, X. R., Giordano, S. J., Yoo, M. and Steggles, A. W. 1995. The isolation and characterization of the human cytochrome $b_5$ gene. Biochem. Biophys. Res. Commun. 209, 894-900. https://doi.org/10.1006/bbrc.1995.1582
  17. Mansouri, A. 1985. Methemoglobinemia. Amer. J. Med. Sci. 289, 200-209. https://doi.org/10.1097/00000441-198505000-00004
  18. Mathews, F. S. 1985. The structure, function and evolution of cytochromes. Prog. Biophys. Mol. Biol. 45, 1-56. https://doi.org/10.1016/0079-6107(85)90004-5
  19. Park, H. C. 2010. Zebrafish (Danio rerio). Mol. Cell. Biol. News 1-11.
  20. Takematsu, H., Kozutsumi, Y., Suzuki, A. and Kawasaki, T. 1992. Molecular cloning of rabbit cytochrome $b_5$ genes: evidence for the occurrence of two separate genes encoding the soluble and microsomal forms. Biochem. Biophys. Res. Commun. 185, 845-851. https://doi.org/10.1016/0006-291X(92)91704-T
  21. Vacaru, A. M., Unlu, G., Spitzner, M., Mione, M., Knapik, E. W. and Sadler, K. C. 2014. In vivo cell biology in zebrafish- providing insights into vertebrate development and disease. J. Cell Sci. 127, 485-495. https://doi.org/10.1242/jcs.140194
  22. Watanabe, F., Nakano, Y., Saido, H., Tamura, Y. and Yamanaka, H. 1992. Cytochrome $b_5$/cytochrome $b_5$ reductase complex in rat liver microsomes has NADH-linked aquacobalamin reductase activity. J. Nutr. 122, 940-944. https://doi.org/10.1093/jn/122.4.940
  23. Wheelock, G. D. and Scott, J. G. 1992. Purification and characterization of cytochrome $b_5$ from the housefly (musca domestica). Comp. Biochem. Physiol. B. Comparative Biochemistry 101, 209-215.
  24. Woods, I. G., Wilson, C., Friedlander, B., Chang, P., Reyes, D. K., Nix, R., Kelly, P. D., Chu, F., Postlethwait, J. H. and Talbot, W. S. 2005. The zebrafish gene map defines ancestral vertebrate chromosomes. Genome Res. 15, 1307-1314. https://doi.org/10.1101/gr.4134305
  25. Xi, Y., Yu, M., Godoy, R., Hatch, G., Poitras, L. and Ekker, M. 2011. Transgenic zebrafish expressing green fluorescent protein in dopaminergic neurons of the ventral diencephalon. Dev. Dyn. 240, 2539-2547. https://doi.org/10.1002/dvdy.22742
  26. Yoo, M. and Steggles, A. W. 1988. The complete nucleotide sequence of human liver cytochrome $b_5$ mRNA. Biochem. Biophys. Res. Commun. 156, 576-580. https://doi.org/10.1016/S0006-291X(88)80881-7
  27. Yoo, M. and Steggles, A. W. 1989. The characterization of three types of partially processed mRNA and two pseudogenes for human liver cytochrome $b_5$. Biochem. Biophys. Res. Commun. 163, 18-24. https://doi.org/10.1016/0006-291X(89)92092-5