Analysis of activation of the Arf locus at various stages of tumorigenesis. (A) Bone marrow cells from Arf^+/+ mice (controls) and Arf^Gfp/Gfp knock-in mice (functionally Arf-null) were transduced with MSCV-Lmo2-IRES-mCherry and transplanted back into lethally irradiated wild-type recipients. Between weeks 4 and 23 after transplantation, individual animals were killed from each cohort of recipients, their thymi removed, and thymocytes analyzed for expression of GFP and mCherry by flow cytometry. Only a small fraction of mCherry⁺ cells expressed GFP by 13 weeks after transplantation. An example of GFP activation in a thymic tumor arising 24 weeks after transplantation is illustrated at the right. GFP-positive cells were also detected in the spleen, bone marrow, and peripheral blood of this mouse. (B) CD4⁻/CD8⁻ double-negative Arf^+/+ and Arf^Gfp/Gfp thymocytes transduced with either MSCV-IRES-mCherry or MSCV-Lmo2-IRES-mCherry were cultured in vitro on OP9-DL1 stromal cells together with IL-7 and FLT-3 ligand. After 20 days in culture, thymocytes were sorted to recover mCherry⁺ cells at the DN2 stage. DN2 mCherry⁺ thymocytes (2 × 10⁵) were transplanted into lethally irradiated mice together with 2 × 10⁵ bone marrow cells. The panels show the cultured thymocytes in the graft before sorting and then from thymi harvested between 6 and 12 weeks after transplantation and analyzed for the presence of mCherry and GFP.