Bone marrow cells were isolated from femurs and tibias of Per2::Luc knock-in mice by flushing with PBS. Freshly isolated LSK CD48CD150+ cells were sorted with a fluorescence-activated cell sorter FACSAria (BD Biosciences) and precultured for 1 day in SF-O3 medium (EIDIA) containing stem cell factor (SCF; 100 ng/ml) (PeproTech) and thrombopoietin (TPO; 100 ng/ml) (PeproTech). The cells were then infected with MLL-AF9–expressing retroviral particles using the Magnetofection technology (OZ Biosciences). After 2 days of infection, green fluorescent protein (GFP)–positive cells were sorted into single cells using FACSAria and cultured for at least six passages to leukemic granulocyte/megakaryocytes progenitor clones. The leukemia cells were subjected to retro-orbital transplantation (0.5 × 106 to 1.0 × 106 cells per mouse) into sublethally irradiated (6 Gy) C57BL/6 J mice to develop AML. Serial transplantations of the leukemia cells caused AML in nonirradiated recipients, which were used for experiments. As controls for the circadian assay, bone marrow cells from Per2::Luc knock-in mice were transplanted into C57BL/6 J mice and used 3 months after transplantation.

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