Abstract
The Ryukyu long-furred rat is an endangered species confined to the southernmost three small islands of Japan (Amami-Oshima, Tokunoshima, and Okinawa). Its population is rapidly decreasing because of roadkill, deforestation, and feral animals. To date, its genomic and biological information are poorly understood. In this study, we successfully immortalized Ryukyu long-furred rat cells by expressing a combination of cell cycle regulators, mutant cyclin-dependent kinase 4 (CDK4R24C) and cyclin D1, together with telomerase reverse transcriptase or an oncogenic protein, the Simian Virus large T antigen. The cell cycle distribution, telomerase enzymatic activity, and karyotype of these two immortalized cell lines were analyzed. The karyotype of the former cell line immortalized with cell cycle regulators and telomerase reverse transcriptase retained the nature of the primary cells, while that of the latter cell line immortalized with the Simian Virus large T antigen had many aberrant chromosomes. These immortalized cells would be valuable for studying the genomics and biology of Ryukyu long-furred rats.
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12 April 2023
A Correction to this paper has been published: https://doi.org/10.1007/s11626-023-00767-1
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Acknowledgements
We would like to thank the Amami Wildlife Conservation Center, Ministry of the Environment (Mr. Shintaro Abe, Dr. Mariko Suzuki, Mr. Yosuke Tsuriya, Ms. Yuna Kimoto, and Ms. Yui Ito) for their essential contributions to sampling. We are also grateful to Dr. Hiroyuki Miyoshi (Riken BioResource Center, present affiliation: Keio University) for providing the lentiviral plasmids and their packaging system. This work was supported by a collaborative research grant from the Wildlife Research Center of the Kyoto University.
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Lanlan Bai, Tohru Kiyono, Miho Inoue-Murayama, and Tomokazu Fukuda conceived, designed, and wrote the manuscript. Lanlan Bai, Noe Kikuchi, Takahiro Eitsuka, Himari Matsusaka, Kiyotaka Nakagawa, Masafumi Katayama, Keiko Ito, Miho Inoue-Murayama, Tohru Kiyono, and Tomokazu Fukuda contributed to data validation and data analysis. All authors read and approved the final manuscript.
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The original version of this article was revised: The given name of a coauthor Tomokazu Fukuda was misspelled (as “Tomkazu“) in this article as originally published.
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11626_2023_757_MOESM1_ESM.pdf
Supplementary file1 No crop agarose gel images of the PCR detection of Fig. 1B. The corresponding areas of agarose gel images were shown by white rectangles. (PDF 1214 KB)
11626_2023_757_MOESM2_ESM.pdf
Supplementary file2 No crop western blot images of protein detection of Fig. 1C. The duplicate detection of anti-tubulin was also shown in this data. The corresponding areas of blot images were shown by black rectangles. (PDF 439 KB)
11626_2023_757_MOESM3_ESM.pdf
Supplementary file3 Reading the A and B values for detecting mycoplasma. The A-value and the B-value of Table 2 have been shown in this data. We measured the B value of primary cells (wild type; WT) four times. This was because the first detection B value was slightly higher than its A value. However, the B values that were detected on the next three occasions were lower than the A value, indicating that the B/A ratio was less than 1 (negative for mycoplasma). (PDF 704 KB)
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Bai, L., Kikuchi, N., Eitsuka, T. et al. Immortalization of primary cells derived from the endangered Ryukyu long-furred rat. In Vitro Cell.Dev.Biol.-Animal 59, 224–233 (2023). https://doi.org/10.1007/s11626-023-00757-3
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DOI: https://doi.org/10.1007/s11626-023-00757-3