Efficient immortalization of cells derived from critically endangered Tsushima leopard cat (Prionailurus bengalensis euptilurus) with expression of mutant CDK4, Cyclin D1, and telomerase reverse transcriptase
- 126 Downloads
Tsushima leopard cat is the subspecies of Amur cats, and it is classified as the most highest class of critically endangered animals. Although the protection activity is highly recognized, the number of animals is decreasing due to the human activity and invasion of domestic cats and infectious disease. In this study, we succeeded primary culture of normal fibroblasts derived from the Tsushima leopard cat (Prionailurus bengalensis euptilurus). Furthermore, we introduced the human derived mutant Cyclin Dependent Kinase 4, Cyclin D1, and telomere reverse transcriptase. We showed that the expression of these three genes efficiently immortalized cells derived from Tsushima leopard cat. Furthermore, we showed that the chromosome pattern of the established cells is identical with the original one. These data indicate that our method of immortalization is useful to establish cell lines from critically endangered cats, which potentially contributes to the re-generation of critically endangered animals from cultured cell with reproductive technique, such as somatic cloning.
KeywordsEndangered animals Immortalization Cellular senescence Tsushima leopard cat
We thank Dr. Hiroyuki Miyoshi (RIKEN, BioResource Center) for providing lentiviral constructs. This work was supported by the Grant from JSPS, KAKENHI, Grant Number 17H03624.
- Associated-Press Rhino in San Diego pregnant, could help save subspecies. https://www.apnews.com/10c94168ec6d4efd894755ac84b17bca/Rhino-in-San-Diego-pregnant,-could-help-save-subspecies. Accessed 2 Jun 2018
- Fukuda T, Iino Y, Eitsuka T et al (2016) 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. https://doi.org/10.1007/s10616-016-0004-0 CrossRefPubMedPubMedCentralGoogle Scholar
- Fukuda T, Eitsuka T, Donai K et al (2018) Expression of human mutant cyclin dependent kinase 4, Cyclin D and telomerase extends the life span but does not immortalize fibroblasts derived from loggerhead sea turtle (Caretta caretta). Sci Rep 8:9229. https://doi.org/10.1038/s41598-018-27271-x CrossRefPubMedPubMedCentralGoogle Scholar
- Izawa M (1991) Ecological study on the two species of Felidae in Japan. In: Maruyama N, Bobek B, Ono Y, Regelin W, Bartos L, Ratcliffe PR (eds) Wildlife conservation. Sankyo, Tokyo, pp 141–143Google Scholar
- Katayama M, Kiyono T, Horie K et al (2016) Establishment of an immortalized cell line derived from the prairie vole via lentivirus-mediated transduction of mutant cyclin-dependent kinase 4, cyclin D, and telomerase reverse transcriptase. Exp Anim 65:87–96. https://doi.org/10.1538/expanim.15-0061 CrossRefPubMedGoogle Scholar
- Katayama M, Hirayama T, Kiyono T et al (2017) Immortalized prairie vole-derived fibroblasts (VMF-K4DTs) can be transformed into pluripotent stem cells and provide a useful tool with which to determine optimal reprogramming conditions. J Reprod Dev 63:311–318. https://doi.org/10.1262/jrd.2016-164 CrossRefPubMedPubMedCentralGoogle Scholar
- Kuroda K, Kiyono T, Isogai E et al (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. https://doi.org/10.1371/journal.pone.0143473 CrossRefPubMedPubMedCentralGoogle Scholar
- Wurster-Hill DH, Doi T, Izawa M, Ono Y (1987) Banded chromosome study of the Iriomote cat. J Hered 78:105–107. https://doi.org/10.1093/oxfordjournals.jhered.a110321 CrossRefPubMedGoogle Scholar