IMMORTALIZATION OF MOUSE BONE MARROW-DERIVED MAST-CELLS WITH AD12-SV40 VIRUS

Mast cells arise in cultures of murine bone marrow in medium supplemen ted with interleukin-3 (IL-3). In the present study, we report the dev elopment of long-term mast cell lines from murine bone-marrow-derived cultured mast cells (BMCMC) following inoculation with adenovirus 12-s imian virus 40 (Ad12-SV40) hybrid virus. One culture of Ad12-SV40 immo rtalized BMCMC (designated as MCP-5) was selected for further analysis . These transformed cells appear similar in morphology and histochemis try to the primary BMCMC from which they are derived and did not shed infectious virus into the culture supernatants. In addition, these cel ls synthesize predominantly chondroitin sulfate proteoglycans and cont ain histamine which is released following a physiologic stimulus. Limi ting-dilution single-cell cloning produced five independent mast cell lines (MCP-5.1 to MCP-5.5). Southern blot analysis of genomic DNA isol ated from these single-cell clones demonstrates different patterns of viral integration in all the five clones. All clones retain responsive ness to an exogenous source of IL-3 for growth and proliferation. Each single-cell clone also demonstrates a unique pattern of cytokine gene expression in response to calcium ionophore A23187 and phorbol-12-myr istate-13-acetate. This suggests that within a culture of BMCMC there are differences in cytokine gene expression that vary from one cell to another. The availability of immortalized mast cell lines derived fro m murine bone marrow which retain their growth factor responsiveness a nd the ability to respond to degranulating stimuli should facilitate f uture studies of mast cell biology.