Generation of Mouse Osteoclastogenic Cell Lines Immortalized with SV40 Large T Antigen

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Abstract
Progress in the field of osteoclast gene regulation has been hampered significantly by the lack of such cell lines. In this study, mouse osteoclast precursor cells were elicited in an osteoclast-inductive coculture system and immortalized using SV40 large T antigen. One of the osteoclast precursor cell lines (MOCP-5) forms 95% tartrate-resistant acid phosphatase positive (TRAP+) multinuclear osteoclast-like cells (OCLs) in the coculture system. The yield of TRAP+ OCLs was 4.5-7x10(4) cells per 10 cm2 dish. Expression of SV40 large T antigen was visualized in the nucleus of MOCP-5 cells and OCLs by immunohistochemistry. MOCP-5 cells were positive for MoMa-2 antigen and nonspecific esterase but negative for F4/80 antigen. OCLs derived from MOCP-5 cells were positive for able to form extensive resorption bone pits on bone slices. The resorbing activity of the OCLs was comparable to that of authentic mouse osteoclasts. Pit formation was inhibited by salmon calcitonin (CT). Acid production by OCLs was demonstrated by vital staining with acridine orange. The OCLs expressed cathepsin K and CT receptors. MOCP-5 cells could be transfected by a construct that carries the beta-galactosidase gene. Transfected MOCP-5 cells expressing beta-galactosidase retain the ability to differentiate into OCLs, indicating a useful model for osteoclast gene regulation. To date, the MOCP-5 cell line has been maintained in continuous culture for 23 months and has maintained the capacity to differentiate into osteoclasts throughout this time. In summary, these data show that a stable immortalized osteoclast precursor cell line has been established and that the immortalization with SV40 large T oncogene does not prevent osteoclast precursor cell differentiation.