Prolonged ex vivo cultivation (13 days) of murine HSCs in combination with gammaretroviral insertional mutagenesis triggers leukemogenesis. (A) Schematic representation of an integrated gammaretroviral LTR-driven SF91-eGFP vector. Indicated are the 5′ and 3′ LTRs consisting of U3, R, and U5 regions. The SFFV promoter is located in both U3 regions and drives the expression of eGFP. (B-D) Representative fluorescence-activated cell sorting analysis for eGFP and B cell–specific B220 expression in leukemic mouse bone marrow (B), spleen (C), and peripheral blood leukocytes (D). (E) Cytospin of bone marrow, (F) histology of spleen, and (G) liver sections showing blasts and infiltrates of leukemic cells (all at 1000× magnification). (H) Blood counts of 5 mice transplanted with the aforementioned leukemia. WBC indicates white cell count; PLT, platelet count; HCT, hematocrit; and RBC, red blood cell count. (I) LM-PCRs on genomic DNA of splenocytes from mice AS1, AS2, AS3, and AS4. Vector integration sites identified by sequencing are indicated. i.c. indicates internal control; and M, marker.