Abstract
A Ujumqin sheep ear marginal tissue (USEM) fibroblast line, frozen in 147 cryovials with 4 × 106 cell each, was successfully established from 33 Ujumqin sheep ear marginal tissues using explant culture and cryopreservation techniques. The cells were morphologically consistent with fibroblasts. The growth curve was typical S-shape and the cell population passed through a lag phase, a logarithmic phase and a plateau phase. The population doubling time (PDT) was approximately 72 h. Tests for bacteria, fungi, viruses and mycoplasma were all negative. Isoenzyme polymorphism indicated that the genetic characteristics of the cell line were stable in vitro. Karyotyping analysis indicated that the chromosome number of a normal cell was of 2n = 54 and 95.4% of the entire population was diploid. The transfection efficiencies of six fluorescent proteins (pEGFP-N3, pEGFP-C1, pDsRed-N1, pEYFP-N1, pECFP-N1 and pECFP-mito) optimal at 48 h were from 18.5% to 30.1%. The cell line met all criteria from the American Type Culture Collection (ATCC). Not only has the germline of this important sheep breed been preserved at the cell level, but also valuable material had been provided for genome, postgenome and somatic cloning research. Moreover, the establishment of this technical platform may provide both technical and theoretical support for storing the genetic resources of other animals and poultry at the cell level.
Similar content being viewed by others
References
Ansari HA, Bosma AA, Broad TE, Bunch TD, Long SE, Maher DW, Pearce PD, Popescu CP (1999) Standard G-, Q-, and R-banded ideograms of the domestic sheep (Ovis aries): homology with cattle (Bos taurus). Report of the committee for the standardization of the sheep karyotype. Cytogenet Cell Genet 87:134–142. doi:10.1159/000015380
Baird GS, Zacharias DA, Tsien RY (2000) Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci USA 97:11984–11989. doi:10.1073/pnas.97.22.11984
Coffin PA, Hall BK (1974) Isozymes of lactate dehydrogenase (LDH) in skeletal tissues of the embryonic and newly hatched chick. J Embryol Exp Morphol 31:169–181
Corley-Smith GE, Brandhorst BP (1999) Preservation of endangered species and populations: a role for genome banking, somatic cell cloning, and androgenesis? Mol Reprod Dev 53:363–367. doi:10.1002/(SICI)1098-2795(199907)53:3<363:AID-MRD12>3.0.CO;2-0
Doyle A, Hay R, Kirsop BE (1990) Animal cells, living resources for biotechnology. Cambridge, New York
Falge R, Ehling C, Niemann H (1996) Preservation of genetic variation in domestic animals using biotechnical methods. Dtsch Tierarztl Wochenschr 103:336–340
Ford CE, Pollock DL, Gustavsson I (1980) Proceedings of the First International Conference for the Standardisation of Banded Karyotypes of Domestic Animals. University of Reading Reading, England. 2nd–6th August 1976. Hereditas 92:145–162
Guan WJ, Ma YH (2003) The construction and evaluation of the cell bank of species of domestic animals on the brink of extinct. Rev China Agric Sci Technol 5:65–70
Guan WJ, Ma YH, Ding H, Yu TY, Zhang HY, Liang HQ (2005) The establ ishment of fibroblast cell line and its biological characteristic research in small tail han sheep. Acta Veterinaria et Zootechnica Sinica 36:511–516
Guan WJ, Liu CQ, Na RS, Lu TF, Ma YH (2006) Research on the six fluorescent protein expression in the fibroblast cells of Mongolia Sheep. Acta Veterinaria et Zootechnica Sinica 37:1141–1148
Hay RI (1992) Cell line preservation and characterization. Oxford, New York
Hodges J (2006) Conservation of genes and culture: historical and contemporary issues. Poult Sci 85:200–209
Kain SR, Adams M, Kondepudi A, Yang TT, Ward WW, Kitts P (1995) Green fluorescent protein as a reporter of gene expression and protein localization. Biotechniques 19:650–655
Kozak RW (1992) Introduction to the issues: recently developed methods for characterizing cell lines. Dev Biol Stand 76:21–24
Lavania UC, Sharma AK (1981) An interphase model for mitotic chromosome organization in eukaryota. Biosystems 14:171–178
Li TJ, Duan XP, Wu QD, Song GM (2002) Study on LDH isoenzymes in tissues of sheep. Anim Sci Vet Med 19:16–17
Liang HQ, Guan WJ, Yue WB (2006) Analysis of chromosomal karyotype of fibroblast cell lines in Menggu Sheep. Chinese Qinghai J Anim Vet Sci 36:9–10
Liu CQ, Guo Y, Guan WJ, Ma YH, Zhang HH, Tang XX (2008) Establishment and biological characteristics of Luxi cattle fibroblast bank. Tissue Cell 40:417–424. doi:10.1016/j.tice.2008.04.005
Ma YH, Wu CX (2001) Evaluation of threatening degree of the animal genetic resources in China. Ecol Domest Anim 22:8–13
Ma YH, Xu GF, Wang DY, Liu HL (2002) Study on dynamic information of animal genetic resources in China. Scientia Agricultura Sinica 35:552–555
Pattison J, Drucker AG, Anderson S (2007) The cost of conserving livestock diversity? Incentive measures and conservation options for maintaining indigenous Pelon pigs in Yucatan, Mexico. Trop Anim Health Prod 39:339–353
Rizzo MA, Springer GH, Granada B, Piston DW (2004) An improved cyan fluorescent protein variant useful for FRET. Nat Biotechnol 22:445–449. doi:10.1038/nbt945
Shaner NC, Patterson GH, Davidson MW (2007) Advances in fluorescent protein technology. J Cell Sci 120:4247–4260. doi:10.1242/jcs.005801
Shen CJ, Guo AL (1980) Chromosomes of Tan-sheep and Mongolia sheep. Chin J Anim Vet Sci 1:83–86
Stacey GN (1997) Standardization in animal cell technology. Folia Microbiol (Praha) 42:113–116
Stacey GN, Masters JR (2008) Cryopreservation and banking of mammalian cell lines. Nat Protoc 3:1981–1989. doi:10.1038/nprot.2008.190
Steube KG, Grunicke D, Drexler HG (1995) Isoenzyme analysis as a rapid method for the examination of the species identity of cell cultures. In Vitro Cell Dev Biol Anim 31:115–119. doi:10.1007/BF02633971
Strober W (2001) Trypan blue exclusion test of cell viability. Curr Protoc Immunol Appendix 3: Appendix 3B. doi: 10.1002/0471142735.ima03bs21
Taberlet P, Valentini A, Rezaei HR, Naderi S, Pompanon F, Negrini R, Ajmone-Marsan P (2008) Are cattle, sheep, and goats endangered species? Mol Ecol 17:275–284. doi:10.1111/j.1365-294X.2007.03475.x
Woelders H, Zuidberg CA, Hiemstra SJ (2006) Animal genetic resources conservation in the Netherlands and Europe: poultry perspective. Poult Sci 85:216–222
Wu CX (1999) The theory and technology of the conservation of animal genetic resources-the specy foundation of animal agricultural continuing development in 21 century. J Yunnan Univ 21:7–10
Wu HM, Guan WJ, Li H, Ma YH (2008) Establishment and characteristics of white ear lobe chicken embryo fibroblast line and expression of six fluorescent proteins in the cells. Cell Bio Int 32:1478–1485. doi:10.1016/j.cellbi.2008.08.006
Xu SC (2003) Studies on activity and isozymes of serum lactate dehydrogenase in Qinghai native goats. J Qinghai Univ 21:1–3
Zeng KW, Chen FY (1997) LDH isoenzyme of vertebrate animals. Harbin, Harbin
Acknowledgments
This research was supported by the “863” National Major Research Program (2006AA10Z198, 2007AA10Z170), National Key Technology R&D Program (2006BAD13B08, 2008BADB2B01) and a project (2008ZX08009-003) from the Ministry of Agriculture of China for transgenic research.
Author information
Authors and Affiliations
Corresponding authors
Additional information
The authors Ri Su Na and Qian Jun Zhao as made the equivalent contribution to this paper.
Rights and permissions
About this article
Cite this article
Na, R.S., Zhao, Q.J., Jin, D.P. et al. Establishment and biological characteristics of Ujumqin sheep fibroblast line. Cytotechnology 62, 43–52 (2010). https://doi.org/10.1007/s10616-010-9260-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10616-010-9260-6