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A new murine osteoblastic cell line immortalized with the SV40 large T antigen

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Abstract

The murine preosteoblastic cell line, MC3T3-E1, is widely used to study bone formation and differentiation in vitro. However, this cell line is unstable in culture. The current study was designed to establish a stable osteoblastic cell line. A mammalian expression vector carrying the SV 40 large T antigen was introduced into a primary culture of cells isolated from the calvaria of newborn mice. Among isolated cell lines, the MN16 cell line was selected for further characterization. The MN16 cell line was cultured for 28 days, and compared with the MC3T3-E1 cell line with or without induction. The expression of bone-related genes was examined using the real-time RT-PCR technique. Alizarin red and von Kossa staining were used to detect mineralization of nodules in the cultures. The cell line showed the characteristics of osteoblastic cells in term of gene expression patterns of various molecular markers and calcium deposition in the cell layer after induction. Furthermore, the MN16 cells showed strong adhesion to the basic domain of collagen, a result that is specific for bone-derived cells. The MN16 cell line was found to be stably differentiated into bone formation cells in vitro and should be useful for studying bone biology.

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Abbreviations

DMEM:

Dulbecco modified Eagle’s medium

α-MEM:

Modified Eagle’s medium

SDS:

Sodium dodecyl sulfate

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Acknowledgments

We thank Dr. M. Watanabe, Mr. S. Imi, Ms A. Yasuda and the staff members of Divisions of Life science Research, Laboratory Animal Science and Radioisotope Research, Department of Research Support, Research Promotion Project, Oita University. This work was supported by Grants-In-Aid for Scientific Research (No. 20390402 to H.Y.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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The authors report no conflicts of interest.

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Authors and Affiliations

  1. Department of Matrix Medicine, Faculty of Medicine, Oita University, 1-1 Hasama-machi, Yufu City, Oita, 879-5593, Japan

    Miki Nakamura-Ota, Hiroyuki Yano, Sawako Adachi, Hideaki Sumiyoshi, Noritaka Matsuo & Hidekatsu Yoshioka

  2. Department of Cell Biology, Faculty of Medicine, Oita University, 1-1 Hasama-machi, Yufu City, Oita, 879-5593, Japan

    Miki Nakamura-Ota & Ryoji Hamanaka

  3. Division of Radioisotope Research, Department of Research Support, Oita University, 1-1 Hasama-machi, Yufu City, Oita, 879-5593, Japan

    Hiroyuki Yano

Authors
  1. Miki Nakamura-Ota
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  2. Ryoji Hamanaka
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  3. Hiroyuki Yano
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  4. Sawako Adachi
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  5. Hideaki Sumiyoshi
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  6. Noritaka Matsuo
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  7. Hidekatsu Yoshioka
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Correspondence to Hidekatsu Yoshioka.

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Nakamura-Ota, M., Hamanaka, R., Yano, H. et al. A new murine osteoblastic cell line immortalized with the SV40 large T antigen. Cell Tissue Bank 15, 373–380 (2014). https://doi.org/10.1007/s10561-013-9394-9

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  • DOI: https://doi.org/10.1007/s10561-013-9394-9

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