Stable expression of a selectable myeloproliferative sarcoma virus in murine T lymphocyte and monocyte cell lines

Abstract

We have investigated whether a retroviral vector based on the myeloproliferative sarcoma virus (MPSV) can be expressed in murine T cells and macrophages. This vector (neo R MPSV) carries the dominant selection marker for neomycin resistance (neo R) and the mos oncogene. The murine T cell line BW5147 and the monocytic cell line P388D1 were either transfected with neo R MPSV DNA or infected with neo R MPSV virus. From both lines, neo R cell clones could be established by retroviral infection, but not by calcium-phosphate precipitationmediated DNA transfection. The efficiency of infection could be increased 60- to 200-fold upon cocultivation of target cells with irradiated neo R MPSV virus-producing cells. All neo R clones showed neo R MPSV specific sequences as revealed by dot blot and Southern blot analysis. The integration and expression of neo R MPSV was stable over a period of now more than 4 months, even in the absence of selection for neomycin resistance. Northern blot analysis showed that neo R clones express full length neo R MPSV. Further, clones of both T cell and monocyte origin were capable to produce infectious virus particles as revealed by focus formation on fibroblasts and conversion of neomycin sensitive fibroblasts to a neomycin resistant phenotype.