Abstract
Lowland Anoa has become endangered due to hunting and human activity. Protection and breeding of endangered species in a controlled environment is the best way of conservation. However, it is not possible to adopt this approach for all endangered species because of the cost involved and the ever-increasing number of critically endangered species. In consideration of these limitations to the conventional conservation methods, we established a primary cell culture of endangered buffalo (Lowland Anoa, Bubalus quarlesi), for the preservation of this biological resource. In addition, we introduced human derived, mutant cyclin dependent kinase 4 (CDK4), Cyclin D, and telomerase reverse transcriptase (TERT) into the primary cells. The successful introduction of these three genes was confirmed by western blot with specific antibodies, and enzymatic activity. We also showed that the expression of mutant CDK4, Cyclin D, and TERT allows us to efficiently establish an immortalized cell line, with an intact chromosome pattern, from Lowland Anoa. To the best of our knowledge, this study is the first investigation that established an immortalized cell line of an endangered wild animal species.
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References
Associated-Press (2009) San Diego’s Frozen Zoo. http://www.cbsnews.com/stories/2002/10/14/tech/main525521.shtml
Burton JA, Hedges S, Burton JA (2005) The taxonomic status, distribution and conservation of the lowland anoa Bubalus depressicornis and mountain anoa Bubalus quarlesi. Mammal Rev 35:25–50. doi:10.1111/j.1365-2907.2005.00048.x
Donai K, Kuroda K, Guo Y, So KH, Sone H, Kobayashi M, Nishimori K, Fukuda T (2013) Establishment of a reporter system to monitor silencing status in induced pluripotent stem cell lines. Anal Biochem 443:104–112. doi:10.1016/j.ab.2013.08.014
Donai K, Kiyono T, Eitsuka T, Guo Y, Kuroda K, Sone H, Isogai E, Fukuda T (2014) Bovine and porcine fibroblasts can be immortalized with intact karyotype by the expression of mutant cyclin dependent kinase 4, cyclin D, and telomerase. J Biotechnol 176:50–57. doi:10.1016/j.jbiotec.2014.02.017
Fukuda T, Mishina Y, Walker MP, DiAugustine RP (2005) Conditional transgenic system for mouse aurora a kinase: degradation by the ubiquitin proteasome pathway controls the level of the transgenic protein. Mol Cell Biol 25:5270–5281
Hayflick L, Moorhead PS (1961) The serial cultivation of human diploid cell strains. Exp Cell Res 25:585–621
IUCN-Species-Survival-Commission (2012) IUCN red list categories and criteria version 3.1. IUCN (International Union for Conservation of Nature)
Kikkawa Y, Yonekawa H, Suzuki H, Amano T (1997) Analysis of genetic diversity of domestic water buffaloes and anoas based on variations in the mitochondrial gene for cytochrome b. Anim Genet 28:195–201. doi:10.1111/j.1365-2052.1997.00101.x
Kuroda K, Kiyono T, Eitsuka T, Isogai H, Takahashi K, Donai K, Isogai E, Fukuda T (2015a) Establishment of cell lines derived from the genus macaca through controlled expression of cell cycle regulators. J Cell Biochem 116:205–211. doi:10.1002/jcb.24963
Kuroda K, Kiyono T, Isogai E, Masuda M, Narita M, Okuno K, Koyanagi Y, Fukuda T (2015b) Immortalization of fetal bovine colon epithelial cells by expression of human cyclin D1, mutant cyclin dependent kinase 4, and telomerase reverse transcriptase: an in vitro model for bacterial infection. PLoS One 10:e0143473. doi:10.1371/journal.pone.0143473
Pedrali-Noy G, Spadari S, Miller-Faures A, Miller AO, Kruppa J, Koch G (1980) Synchronization of HeLa cell cultures by inhibition of DNA polymerase alpha with aphidicolin. Nucleic Acids Res 8:377–387
Qin XY, Fukuda T, Yang L, Zaha H, Akanuma H, Zeng Q, Yoshinaga J, Sone H (2012) Effects of bisphenol A exposure on the proliferation and senescence of normal human mammary epithelial cells. Cancer Biol Ther 13:296–306. doi:10.4161/cbt.18942
Shiomi K, Kiyono T, Okamura K, Uezumi M, Goto Y, Yasumoto S, Shimizu S, Hashimoto N (2011) CDK4 and cyclin D1 allow human myogenic cells to recapture growth property without compromising differentiation potential. Gene Ther 18:857–866. doi:10.1038/gt.2011.44
Takahashi A, Ohtani N, Yamakoshi K, Iida S, Tahara H, Nakayama K, Nakayama KI, Ide T, Saya H, Hara E (2006) Mitogenic signalling and the p16INK4a-Rb pathway cooperate to enforce irreversible cellular senescence. Nat Cell Biol 8:1291–1297. doi:10.1038/ncb1491
Acknowledgments
We thank Dr. Hiroyuki Miyoshi (RIKEN, BioResource Center) for providing lentiviral constructs. This work was supported by the research grant, JSPS KAKENHI.
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Tomokazu Fukuda and Yuuka Iino have contributed equally to this work.
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Fukuda, T., Iino, Y., Eitsuka, T. et al. Cellular conservation of endangered midget buffalo (Lowland Anoa, Bubalus quarlesi) by establishment of primary cultured cell, and its immortalization with expression of cell cycle regulators. Cytotechnology 68, 1937–1947 (2016). https://doi.org/10.1007/s10616-016-0004-0
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DOI: https://doi.org/10.1007/s10616-016-0004-0