title: Bovine and porcine fibroblasts can be immortalized with intact karyotype by the expression of mutant cyclin dependent kinase 4, cyclin D, and telomerase.
abstract: Cattle and pigs comprise the most economically important livestock. Despite their importance, cultured cells from these species, which are useful for physiological analyses, are quite limited in cell banks. One of the reasons for the limited number of cell lines is the difficulty in their establishment. To overcome limitations in cell-line establishment, we attempted to immortalize bovine and porcine fibroblasts by transduction of multiple cell cycle regulators (mutant cyclin dependent kinase 4, cyclin D and telomerase reverse transcriptase). The transduced cells continued to display a stable proliferation rate and did not show cellular senescence. Furthermore, cell cycle assays showed that induction of these exogenous genes enhanced turnover of the cell cycle, especially at the G1-S phase. Furthermore, our established cell lines maintained normal diploid karyotypes at 98-100%. Our study demonstrated that bypassing p16/Rb-mediated cell arrest and activation of telomerase activity enabled efficient establishment of immortalized bovine- and porcine-derived fibroblasts. The high efficiency of establishing cell lines suggests that the networks of cell cycle regulators, especially p16/Rb-associated cell cycle arrest, have been conserved during evolution of humans, cattle, and pigs.
authors.affiliation: Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-amamiyamachi, Aoba-ku, Sendai 981-8555, Japan. Electronic address: studiotwentyseven@gmail.com.
authors.forename: Kenichiro
authors.initials: K
authors.lastname: Donai
authors.affiliation: Division of Virology, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan. Electronic address: tkiyono@ncc.go.jp.
authors.forename: Tohru
authors.initials: T
authors.lastname: Kiyono
authors.affiliation: Faculty of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1, Higashijima, Akiha-ku, Niigata 956-8603, Japan. Electronic address: eitsuka@nupals.ac.jp.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Eitsuka
authors.affiliation: Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-amamiyamachi, Aoba-ku, Sendai 981-8555, Japan. Electronic address: yijie_guo@163.com.
authors.forename: Yijie
authors.initials: Y
authors.lastname: Guo
authors.affiliation: Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-amamiyamachi, Aoba-ku, Sendai 981-8555, Japan. Electronic address: kengodoubi@gmail.com.
authors.forename: Kengo
authors.initials: K
authors.lastname: Kuroda
authors.affiliation: Environmental Exposure Research Section, Center for Environmental Risk Research, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan. Electronic address: hsone@nies.go.jp.
authors.forename: Hideko
authors.initials: H
authors.lastname: Sone
authors.affiliation: Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-amamiyamachi, Aoba-ku, Sendai 981-8555, Japan. Electronic address: emiko@bios.tohoku.ac.jp.
authors.forename: Emiko
authors.initials: E
authors.lastname: Isogai
authors.affiliation: Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-amamiyamachi, Aoba-ku, Sendai 981-8555, Japan. Electronic address: tomofukuda@bios.tohoku.ac.jp.
authors.forename: Tomokazu
authors.initials: T
authors.lastname: Fukuda
mesh_terms: D000818:Animals; D002417:Cattle; D002453:Cell Cycle; D002460:Cell Line; D049109:Cell Proliferation; D056741:Cyclin D / Q000235:genetics / Q000378:metabolism*; D051358:Cyclin-Dependent Kinase 4 / Q000235:genetics / Q000378:metabolism*; D004171:Diploidy; D004622:Embryo, Mammalian / Q000166:cytology*; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D059785:Karyotype; D013552:Swine; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D014161:Transduction, Genetic
chemical_list: D056741:Cyclin D; D051358:Cyclin-Dependent Kinase 4; C509186:TERT protein, human; D019098:Telomerase
keywords: Cell cycle; Cellular senescence; Fibroblasts; Immortalization; Livestock