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Zebrafish에서 인간 KCNE1 유전자 발현에 관한 연구

Expression of Human KCNE1 Gene in Zebrafish

  • 박현정 (계명대학교 생명과학전공) ;
  • 유민 (계명대학교 생명과학전공)
  • Park, Hyeon Jeong (Department of Biological Sciences, Keimyung University) ;
  • Yoo, Min (Department of Biological Sciences, Keimyung University)
  • 투고 : 2017.01.19
  • 심사 : 2017.01.31
  • 발행 : 2017.05.30

초록

본 연구에서는 zebrafish에 인간의 KCNE1 유전자가 삽입된 형광단백질 vector를 microinjection하고, 그 발현 여부를 확인하고자 하였다. 먼저 양 말단에 제한효소(EcoRΙ, BamHΙ) site를 넣어 제작한 primer들로 genomic DNA에서 KCNE1 유전자를 분리하였다. 그 결과는 약 402 bp 크기의 DNA band였고 이 PCR 산물을 형광단백질 vector인 pPB-CMVp-EF1-GreenPuro 속에 클로닝하여 pPB-CMVp-hKCNE1-EF1-GreenPuro plasmid를 제작하였다. 이렇게 준비된 형광 vector를 zebrafish 수정란에 microinjection하였고, 부화된 치어에서 RT-PCR과 DNA sequencing을 통해 GFP 및 hKCNE1의 발현을 최종 확인하였다. 본 연구는 향후 QT 연장증후군(LQTs)에 대한 동물 모델로써 신경자극 전도, 유전자 치료, 유용 유전자 클로닝을 위한 기술 개발에 응용될 수 있을 것으로 기대된다.

This study was aimed to produce a transgenic zebrafish expressing the human KCNE1 gene. Initially, the entire CDS of the human KCNE1 gene was amplified from a human genomic DNA sample by polymerase chain reaction using a primer set engineered with restriction enzyme sites (EcoRI, BamHI) at the 5' end of each primer. The resultant 402 bp KCNE1 amplicon flanked by EcoR1 and BamH1 was obtained and subsequently cloned into a plasmid vector pPB-CMVp-EF1-GreenPuro. The integrity of the cloned CDS sequence was confirmed by DNA sequencing analysis. Next, the recombinant vector containing the human KCNE1 (pPB-CMVp-hKCNE1-EF1-GreenPuro) was introduced into fertilized eggs of zebrafish by microinjection. Successful expression of the recombinant vector in the eggs was confirmed by the expression of the fluorescence protein encoded in the vector. Finally, in order to assure that the stable expression of the human KCNE1 gene occurred in the transgenic animal, RNAs were extracted from the animal and the presence of KCNE1 transcripts was confirmed by RT-PCT as well as DNA sequencing analysis. The study provides a methodology to construct a useful transgenic animal model applicable to the development of diagnostic technologies for gene therapy of LQTS (Long QT Syndrome) as well as tools for cloning of useful genes in fish.

키워드

참고문헌

  1. Amsterdam, A., Burgess, S., Golling, G., Chen, W., Sun, Z., Townsend, K., Farrington, S., Haldi, M. and Hopkins, N. 1999. A large-scale insertional mutagenesis screen in zebrafish. Genes Dev. 13, 2713-2724. https://doi.org/10.1101/gad.13.20.2713
  2. Axelrod, H. R. and Schulz, L. P. 1955. A handbook of tropical aquarium fishes. McGraw-Hill Book co. 271.
  3. Bhimachar, B. S. and Subra Rau, A. 1941. The Fishes of Mysore State. I. fishes of Kadur District. Half-yearly J. 1, 141-153.
  4. Busch-Nentwich, E., Sollner, C., Roehl, H. and Nicolson, T. 2004. The deafness gene dfna5 is crucial for ugdh expression and HA production in the developing ear in zebrafish. Development 131, 943-951. https://doi.org/10.1242/dev.00961
  5. Cho S. W., Park, H. J., Kim, G. Y., Nam, M. K., Kim, H. Y. Ko, I., Kim, C. H. and Rhim, H. 2006. Establishment of the expression system of human HtrA2 in the zebrafish. J. Life Sci. 16, 571-578. https://doi.org/10.5352/JLS.2006.16.4.571
  6. Chrenek, P., Chrastinova, L., Kirchnerova, K., Makarevich, A. V. and Foltys, V. 2007. The yield and composition of milk from transgenic rabbits. Asian-Australasian J. Anim. Sci. 20, 482-486. https://doi.org/10.5713/ajas.2007.482
  7. Ding, S., Wu, X., Li, G., Han, M., Zhuang, Y. and Xu, T. 2005. Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice. Cell 122, 473-483. https://doi.org/10.1016/j.cell.2005.07.013
  8. Driever, W., Stemple, D., Schier, A. and Solnica-Krezel, L. 1994. Zebrafish: genetic tools for studying vertebrate development. Trends Genet. 10, 152-159. https://doi.org/10.1016/0168-9525(94)90091-4
  9. Geelen, J. L., Doevendans, P. A., Jongbloed, R. J. E., Wellens, H. J. J. and Geraedts, J. P. M. 1998. Molecular genetics of inherited long QT syndromes. Eur. Heart J. 19, 1427-1433. https://doi.org/10.1053/euhj.1998.0873
  10. Gibbs, P. D. and Schmale, M. C. 2000. GFP as a genetic marker scorable throughout the life cycle of transgenic zebrafish. Biotechnology 2, 107-125.
  11. Hirao, H., Shimizu, W., Kurita, T., Suyama, K., Aihara, N., Kamakura, S. and Shimomura, K. 1996. Frequency-dependent electrophysiologic properties of ventricular repolarization in patients with congenital long QT syndrome. J. Amer. Coll Cardiol. 28, 1269-1277. https://doi.org/10.1016/S0735-1097(96)00313-0
  12. Hisaoka, K. K. and Firlit, C. F. 1962. The embryology of the blue gourami, Trichogaster trichopterus (Pallas). J. Mor. 3, 239-253.
  13. Huang, H., Ju, B., Lee, K. Y. and Lin, S. 2004. The zebrafish: genetics, genomics and informatics. Methods Cell Biol. 77, 403-411.
  14. Jackman, W., Friday, K. and Anderson. 1988. The long QT syndromes: A critical review, new clinical observations and a unifying hypothesis. Prog Cardiovasc Dis. 31, 115-172. https://doi.org/10.1016/0033-0620(88)90014-X
  15. Kato, M., Yamanouchi, K., Ikawa, M., Okabe, M., Naito, K. and Tojo, H. 1999. Efficient selection of transgenic mouse embryos using EGFP as a marker gene. Mol. Reprod. 54, 43-48. https://doi.org/10.1002/(SICI)1098-2795(199909)54:1<43::AID-MRD6>3.0.CO;2-N
  16. Keating, M. T. 1996. The long QT syndrome. A review of recent molecular genetic and physiologic discoveries. Medicine 75, 1-5. https://doi.org/10.1097/00005792-199601000-00001
  17. Koo, S. H., Woom, F. H. and Demund, J. D. L. 2006. Genetic polymorphisms in KCNE1, HERG, KCNE1 and KCNE2 genes in the Chises, Malay and Indian populations of Singapore. Br. J. Clin Pharmacol. 61, 301-308. https://doi.org/10.1111/j.1365-2125.2005.02545.x
  18. Park, J. K., Jeon, I. S., Lee, Y. K., Lee, P. Y., Kim, S. W., Kim, S. J., Lee, H. G., Han, J. H. and Park, C. G. 2003. Increased of the red blood cell in peripheral plasma of transgenic pigs harboring hEPO gene. Reprod. Dev. Biol. 27, 317-24.
  19. Patton, E. E. and Zon, L. I. 2001. The art and design of genetic screens: zebrafish. Nat. Rev. Genet. 2, 956-966. https://doi.org/10.1038/35103567
  20. Splawski, I., Jiaxiang, S., Katherine, W. T., Michael, G. V., Michael, H. L. and Mark, T. K. 1998. Genomic structure of three long QT syndrome genes: KVLQT1, HERG, and KCNE1. Genomics 51, 86-97. https://doi.org/10.1006/geno.1998.5361
  21. Viskin, S., Alla, S. R. and Barron, H. V. 1996. Mode of onset of torsade de pointes in congenital long QT syndrome. J. Amer. Coll Cardiol. 28, 1262. https://doi.org/10.1016/S0735-1097(96)00311-7
  22. Yeo, S. I., Kim, S. W., Kim, Y. N., You, K. H., Shin, S. W., Kim, M. H., Song, J. C. and Yoo, M. 2002. Complete nucleotide sequence of KCNE1 in Korean genome. J. Exp. Biomed. Sci. 8, 185-188.