Establishment and Identification of a Debao Pony Ear Marginal Tissue Fibroblast Cell Line

  • Zhou, X.M. (Institute of Animal Sciences, Chinese Academy of Agricultural Sciences) ;
  • Ma, Y.H. (Institute of Animal Sciences, Chinese Academy of Agricultural Sciences) ;
  • Guan, W.J. (Institute of Animal Sciences, Chinese Academy of Agricultural Sciences) ;
  • Zhao, D.M. (College of Veterinary Medicine, China Agricultural University)
  • Received : 2003.12.15
  • Accepted : 2004.05.21
  • Published : 2004.10.01


The Debao pony ear marginal tissue fibroblast cell line (NDPEM 2/2) was uccessfully established using either primary explant technique or collagenase technique. The characterizations of the cell line were identified as following: the cells were adherent and of density limitation; population doubling time (PDT) of cells made with the two techniques were 35.9 h and 48 h, respectively; chromosome analysis showed that the frequency of cell chromosome number to be 2n=64 was 91.3%-92.8%. Confirmed by isoenzyme analysis, this cell line had no cross- contamination. Tests for microbial contamination from bacteria, fungi, virus or mycoplasma were negative. This newly established cell line meets all the standard quality controls of ATCC. It will provide a precious genetic resource for the conservation of the Debao pony breed, as well as effective experimental material for genetic studies on Debao ponies.


Supported by : National Science Foundation of China


  1. Barile, M. E. and S. Rottem. 1993. Mycoplasmas in cell culture. In: Rapid diagnosis of mycoplasmas (Ed. L. Kahane and A. Adoni). Plenum Press, New York. pp. 155-193.
  2. Bowling, A. T., M. Breen, B. P. Chowdhary, K. Hirota, T. Lear, L. V. Millon, F. A. Ponce de Leon, T. Raudsepp and G. Stranzinger. 1997. International system for cytogenetic nomenclature of the domestic horse: 1. Report of the Third International Committee for the Standardization of the Domestic Horse Karyotype, Davis, CA, USA, 1996. Chromosome Research 5:433-443.
  3. Butler, M. 1999. Cell counting and viability measurements. In: Animal Cell Biotechnology Methods and Protocols (Ed. Nigel Jenkins). Humana Press Inc., New Jersey. pp. 131-144.
  4. Committee on Standardized Genetic Nomenclature for Mice. 1972. Standard karyotype of the mouse Mus musculis. J. Hered. 63:69.
  5. Doyle, A., C. Morris and J. M. Mowles. 1990. Quality control. In:Animal cells, living resources for biotechnology. (Ed. A. Doyle,R. Hay and B. E. Kirsop). Cambridge University Press,Cambridge, U.K.
  6. E, Zh. 1994. Tests for biological characteristics in cell cultures. In:Tissue culture and molecular cytology technique (Ed. E. Zh)Beijing press, Beijing. p. 155.
  7. Editing Committee of Report on Domestic Animal Genetic Resources in China. 2004. Report on Domestic Animal Genetic Resources in China. China Agricultural press, Beijing.
  8. Editing Section of Horse and Ass Breeds in China. 1986. Horse and Ass Breeds in China. Shanghai Scientific and Technical Publishers, Shanghai.
  9. Ford, C. E., D. L. Pollock and I. Gustavsson. 1980. Proceedings of the First International Conference for the Standardization of Banded Karyotypes of Domestic Animals. Hereditas 92:145-162.
  10. Freshney, R. I. 2000. Culture of animal cells: A manual of basic technique. 4th ed. Wiley-Liss, New York.
  11. Garner, C. M., L. M. Hubbold and P. R. Chakraborti. 2000. Mycoplasma detection in cell cultures: a comparison of four methods. Br. J. Biomed. Sci. 57:295-301.
  12. Harnden, D. G. 1974. Skin culture and solid tumor technique. In: Human chromosome methodology. 2nd edn (Ed. J. J. Yunis). Academic Press, INC., New York and London.
  13. Hay, R. I. 1992. Cell line preservation and characterization. In: Animal cell culture: a practical approach. 2nd edn (Ed. R. I. Freshney). Oxford University Press, Oxford. pp. 104-135.
  14. Hay, R. J. 1978. Preservation of cell culture stocks in liquid nitrogen. TCA Manual 4:787-790.
  15. Ho, S. K., E. E. Lister and S. P. Touchbum. 1997. An Overview of Canadian farm animal genetic resources conservation and its associated biotechnological approaches. In: Proceedings of international conference of animal biotechnology. 155-160.
  16. Lavappa, K. S. 1978. Survey of ATCC stocks of human cell lines for Hela contamination. In vitro 14(5):469-475.
  17. Lee, C. K. and J. A. Piedrahita. 2003. Transgenesis and germ cell engineering in domestic animals. Asian-Aust. J. Anim. Sci.16(6):910-927.
  18. Masover, G. K. and F. A. Becker. 1998. Detection of mycoplasmas in cell cultures by cultural methods. In: Methods in molecular biology. Vol. 104: Mycoplasma protocols (Ed. R. J. Miles and R. A. J. Nicholas). Humana Press Inc., Totawa NJ. pp. 207-15, 217-26
  19. Nelson, W. A., D. Daniels and R. R. Flandermeyer. 1981. Crosscontamination of cells in culture. Sci. 212:446-452.
  20. Nelson-Rees, W. and R. R. Flandermeyer. 1977. Inter- and intraspecies contamination of human breast tumor cell lines HBC and BrCa5 and other cell cultures. Sci. 195:1343-1344.
  21. Nims, R. W., A. P. Shoemaker, M. A. Bauternschub, L. J. Rec and J. W. Harbell. 1998. Sensitivity of isoenzyme analysis for the detection of interspecies cell line cross-contamination. In vitro Cell Dev. Biol. Anim. 34:35-39.
  22. O'Brien, S. U., G. Kleiner, R. Olson and J. E. Shannon. 1977. Enzyme polymorphisms as genetic signatures in human cells cultures. Sci. 195:1345-1348.
  23. Oishi, T. 1997. Conservation and evaluation of animal genetic resources. Farming Japan, Vol. 31-6:18-25
  24. Parodi, B., O. Aresu, D. Bini, R. Lorenzini, F. Schena, P. Visconti, M. Cesaro, D. Ferrera, V. Andreotti and T.Ruzzon. 2002. Species identification and confirmation of human and animal cell lines: a PCR-based method. Biotechniques 32(2):432-440.
  25. Ren F. L., Y. Li and Y. Zhang. 2002. In vitro cultivation and freezing of bovine skin fibroblast cells. J. Yellow Cattle Sci. 28:8-10.
  26. Ryder, O. A. and K. Benirschke. 1997. The potential use of 'cloning' in the conservation effort. Zoo Biology, 16:295-300.
  27. Shepel, L. A., L. W. Morrissey, L. C. Hsu and M. N. Could. 1994. Bivariate flow karyotyping, sortiing, and peak assignment of all rat chromosomes. Genomics 19:75-85.
  28. Shi, L. M. 1989. Freezing zoo: 1. Establishment and application of wild animal cell banks. Biology Bulletin 6:1-3.
  29. Simon, D. L. 1999. Better Decisions in conservation of farm animal genetic resources by use of international sources of information. Information in Decision Making in Agricultural Research and Practice/IAALD QB XLIV, 1/2.
  30. Uphoff, C. C. and H. G. Drexler. 2002. Comparative PCR analysis for detection of mycoplasma infections in continuous cell lines. In vitro Cell Dev. Biol. Anim. 38:79-85.
  31. Wu, Ch. X. 1999. Theory and Technique of Animal genetic resources conservation. 1. Idioplasmic basis for persist development of animal agriculture at 21 century. J. Yun Nan University 21:7-10.
  32. Xiao, Y. P. 2001. In vitro culture of connective tissue. In: The principle and technique of in vitro culture (Ed. Q. SH. Xue). Science Press, Beijing. pp. 436-452.
  33. Yang, T. X. and Y. W. Wu. 1999. The application of electrophoresis technique in agriculture sciences. In: Electrophoresis. 2nd edn (Ed. ZH. X. He and SH. ZH. Zhang). Science press, Beijing. pp. 258-308.

Cited by

  1. Establishment and characterization of a fibroblast cell line from the Mongolian horse vol.45, pp.7, 2009,
  2. Establishment and characterization of a fibroblast cell line derived from Texel sheep vol.87, pp.3, 2009,
  3. ) vol.8, pp.2, 2010,
  4. Inhibitory effects of Vitamin E on UVB-induced apoptosis of chicken embryonic fibroblasts vol.35, pp.4, 2011,
  5. Establishment and characterization of a fibroblast cell line derived from Mongolian sheep vol.82, pp.2, 2011,
  6. Establishment and characteristics of Iranian Sistani cattle fibroblast bank: a way to genetic conservation vol.9, pp.2, 2017,