Ectopic expression of FOG-1 in MCPs blocks mast cell maturation and enables alternate lineage development. (A) FACS analysis for TER119 and FcεRI expression of clonal primary BMMCs retrovirally transduced with empty vector or FOG-1–IRES-eGFP–expressing retroviruses. GFP⁺ cells were sorted 2 d after retroviral infection and cultured in EPO, TPO, IL-3, and SCF for 6 d. (B) Scheme for isolation and retroviral infection of day 2 MCPs. (C) May-Grunwald-Giemsa stains of day 2 MCPs transduced with empty vector, FOG-1, or the FOG-1 mutants m1,5,6,9, ΔN67, and ΔN67, mCTBP expressing retroviruses; cultured in mast cell media containing EPO, TPO, SCF, and IL-3 for 4 d; sorted for GFP expression (GFP⁺ cells); and cultured for an additional of 5 d in mast cell media containing EPO, TPO, SCF, and IL-3. Boxes indicate cells seen in additional fields. Bars, 10 μm. (D) RT-PCR analysis for erythroid, megakaryocytic, and mast cell marker gene expression in the cells shown in C (harvested 5 d after sorting GFP⁺ cells). (E) Quantitative RT-PCR analysis for FOG-1 mRNA transcript levels in day 2 MCPs retrovirally transduced with the empty vector or FOG-1, sorted for GFP⁺ expression after 4 d, and cultured for an additional 5 d in the presence of SCF, IL-3, EPO, and TPO. Levels are displayed relative to those determined in parallel from sorted mouse MEPs and normalized to GAPDH mRNA transcript levels. The relative signal from FOG-1^−/− cells (reference 10) is shown as an indicator of experimental background. All measurements were made in triplicate. The error bars represent the SEM.