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

Mast cells arise in cultures of murine bone marrow in medium supplemented with interleukin-3 (IL-3). In the present study, we report the development of long-term mast cell lines from murine bone-marrow-derived cultured mast cells (BMCMC) following inoculation with adenovirus 12-simian virus 40 (Ad12-SV40) hybrid virus. One culture of Ad12-SV40 immortalized BMCMC (designated as MCP-5) was selected for further analysis. These transformed cells appear similar in morphology and histochemistry to the primary BMCMC from which they are derived and did not shed infectious virus into the culture supernatants. In addition, these cells synthesize predominantly chondroitin sulfate proteoglycans and contain histamine which is released following a physiologic stimulus. Limiting-dilution single-cell cloning produced five independent mast cell lines (MCP-5.1 to MCP-5.5). Southern blot analysis of genomic DNA isolated from these single-cell clones demonstrates different patterns of viral integration in all the five clones. All clones retain responsiveness 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-myristate-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 from murine bone marrow which retain their growth factor responsiveness and the ability to respond to degranulating stimuli should facilitate future studies of mast cell biology.