Korean Journal of Ichthyology (한국어류학회지)

The Ichthyological Society of Korea (한국어류학회)
권호 표지

Establishment and Characterization of Clonal Cell Lines from Zebrafish, Danio rerio

제브라피쉬(Danio rerio) 배아로부터 동형세포주 확립

  • Lee, Ki-Young (School of Marine Life Science, College of Ocean Science & Technology, Kunsan National University)
  • 이기영 (군산대학교 해양과학대학 해양생명과학부)
  • Received : 2008.02.14
  • Accepted : 2008.02.28
  • Published : 2008.03.31
Download PDF
⟨ Previous Next ⟩

Abstract

Three types of clonal cell lines were isolated according to their size and phenotype from the adherent cell populations in long-term liquid cultures from the embryonic fibroblast cells of Zebrafish, Danio rerio. All kind of cell lines were well proliferated. The size and number of clonal cell lines derived colonies from stable embryonic cells were significantly increased in the presence of NAC and A2P conditioned medium from the cell lines. The stable cell lines and clonal cell lines were cap-able of well proliferation in vitro. These cell lines have been maintained in continuous culture without change in characteristics. A majority of the clonal cells (80%) was shown a normal chromosomal complement (50 chromosomes, 2N) in according with FACs analysis. Majority of cells were positive to vimentin staining and none of them were positive for nestin and Oct -4 by immunocytochemistry. These results indicate that the clonal cell lines obtained from cultured cells are fibroblasts and may be extremely useful in genetic manipulation for further nuclear transfer and fish cloning.

제브라피쉬 배아로부터 확보한 세포주로부터 외부형태와 세포 크기에 따라 3종류의 동형세포주를 확립하였다. 활발하게 증식하는 안정된 세포주 및 이들로부터 확립된 동형세포주의 세포특성은 변하지 않고 지속적으로 유지되었으며, 안정된 세포주로부터 총 18개의 콜로니를 확보하여 배양한 다음 3종류의 동형세포주를 선별하여 세포 특성을 분석하였다. 대부분의 동형세포주는 약 80% 정도 정상적인 염색체(2N=50)를 가지고 있었으며 FACs 분석과 일치하였다. 배아로부터 확립된 동형세포주에 항체테스트 결과, vimentin에서 양성을 보이는 결과로 볼 때 확립된 세포주는 분화된 섬유세포임이 확인되었다. 이러한 결과는, 확립된 동형세포주를 이용한 유전자조작과 어류복제에의 활용도를 높일 수 있음을 시사한다.

Keywords

Acknowledgement

Supported by : Korea Science and Engineering Foundation (KOSEF), Kunsan National University

References

  1. Amsterdam, A., S. Burgess, G. Golling, W. Chen, Z. Sun, K. Townsend, S. Farrington, M. Haldi and N. Hopkins. 1999. A large-scale insertional mutagenesis screen in zebrafish, Genes Dev., 13: 2713-2724 https://doi.org/10.1101/gad.13.20.2713
  2. Bradford, C.S., L. Sun and D.W. Barnes. 1994. Basic fibroblast growth factor stimulates proliferation and sup-presses melanogenesis in cell cultures derived from early zebrafish embryos. Mol. Mar. Biol. Biotechnol., 3: 78-86
  3. Cibelli, J.B., S.L. Stice, P.J. Golueke, J.J. Kane, J. Jerry, C. Blackwell, F.A. Ponce de Leon and J.M. Robl. 1998. Cloned transjenic calves produced from nonquiescent fetal fibroblasts. Science, 280: 1256-1258 https://doi.org/10.1126/science.280.5367.1256
  4. Collodi, P. and D.W. Barnes. 1990. Mitogenic activity from trout embryos. Proc. Natl. Acad. Sci. U.S.A., 87: 3498-502
  5. Collodi, P., Y. Kamei, T. Ernst, C. Miranda, D.R. Buhler and D.W. P. Barnes. 1992. Culture of cells from zebrafish (Brachydanio rerio) embryo and adult tissues. Cell. Biol. Toxicol., 8: 43-61 https://doi.org/10.1007/BF00119294
  6. Driever, W. and Z. Rangini 1993. Characterization of a cell line derived from zebrafish (Brachydanio rerio) embryos. In Vitro Cell. Dev. Biol. Anim., 29: 749-754 https://doi.org/10.1007/BF02631432
  7. Endo, A. and Ingalls. 1968. Chromosomes of the zebra fish. A model for cytogenetic, embryologic, and ecologic study. J. Hered., 59: 382-384 https://doi.org/10.1093/oxfordjournals.jhered.a107755
  8. Fan, L., J. Crodian and P. Collodi. 2004. Culture of embryonic stem cell lines from zebrafish. Methods Cell Biol., 76: 151-160 https://doi.org/10.1016/S0091-679X(04)76009-4
  9. Ghosh, C. and P. Collodi 1994. Culture of cells from zebrafish (Brachydanio rerio) blastula-stage embryos. Cytotechnology, 14: 21-26 https://doi.org/10.1007/BF00772192
  10. Haffter, P., M. Granato, M. Brand, M.C. Mullins, M. Hammerschmidt, D.A. Kane, J. Odenthal, F.J. van Eeden, Y.J. Jiang and C.P. Heisenberg. 1996. The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio. Development, 123: 1-36
  11. Kasinathan, P., J.G. Knott, Z. Wang, D.J. Jerry and J.M. Robl. 2001. Production of calves from G1 fibroblasts. Nat Biotechnol., 19: 1176-1178 https://doi.org/10.1038/nbt1201-1176
  12. Lai, L., D. Kolber-Simonds, K.W. Park, H.T. Cheong, J.L. Greenstein, G.S. Im, M. Samuel, A. Bonk, A. Rieke, B.N. Day, C.M. Murphy, D.B. Carter, R.J. Hawley and R.S. Prather. 2002. Production of {alpha}-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science, 295: 1089-1092 https://doi.org/10.1126/science.1068228
  13. Lee, K.Y., H. Huang, B. Ju, Z. Yang and S. Lin. 2002. Cloned zebrafish by nuclear transfer from long-term-cultured cells. Nat. Biotechnol., 20: 795-799 https://doi.org/10.1038/nbt721
  14. Lin, T.M., J.L. Tsai, S.D. Lin, C.S. Lai and C.C. Chang. 2005. Accelerated growth and prolonged lifespan of adipose tissue-derived human mesenchymal stem cells in a medium using reduced calcium and antioxidants. Stem Cells Dev., 14: 92-102 https://doi.org/10.1089/scd.2005.14.92
  15. Ma, C., L. Fan, R. Ganassin, N. Bols and P. Collodi. 2001. Production of zebrafish germ-line chimeras from embryo cell cultures. Proc Natl. Acad. Sci., U. S. A. 98: 2461-246
  16. McCreath, K.J., J. Howcroft, K.H. Campbell, A. Colman, A.E. Schnieke and A.J. Kind. 2000. Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature, 405: 1066-1069 https://doi.org/10.1038/35016604
  17. Murphey, R.D. and L.I. Zon. 2002. Attack of the fish clones. Nat Biotechnol., 20: 785-786 https://doi.org/10.1038/nbt0802-785
  18. Sun, L., C.S. Bradford, C. Ghosh, P. Collodi and D.W. Barnes. 1995. ES-like cell cultures derived from early zebrafish embryos. Mol. Mar. Biol. Biotechnol., 4: 193-199
  19. Wilmut, I., A.E. Schnieke, J. McWhir, A.J. Kind and K.H. Campbell. 1997. Viable offspring derived from fetal and adult mammalian cells. Nature, 385: 810-813 https://doi.org/10.1038/385810a0