Mll-Af4 and mutant N-Ras^G12Dcooperate to generate an aggressive and serially transplantable B-ALL. (A) Strategy for generation of cells harboring both Mll-Af4 and mutant N-Ras by retroviral transduction. (B) Kaplan-Meier survival curves of primary, secondary, and tertiary leukemias. LSKs from 2 independent Mll-Af4 donor mice were transformed (n ≥ 5 for each donor). Mll-Af4/MIG controls did not develop disease 160 to 200 days after transplant when mice were euthanized and assessed for leukemic involvement with ≤5% detectable leukemic cells in peripheral blood or bone marrow at time of euthanization (data not shown). Secondary and tertiary leukemias were generated from injection of 100 000 primary leukemia cells (n ≥ 4 for all groups). (C) Pro-B cell (B220⁺Cd43⁺IgM⁻) immunophenotype of primary leukemias. (D) Histology (hematoxylin and eosin stain) of bone marrow (i), spleen (ii), liver (iii), and central nervous system (iv) from primary B-ALLs (scale bars, 200 μm). Insets show higher-magnification views (scale bars, 50 μm). (E) Spleen weights in primary (n = 10), secondary (n = 26), and tertiary (n = 19) Mll-Af4/N-Ras^G12D leukemic mice from ≥3 independent transplant experiments generated from 3 independent Mll-Af4 donors. Spleens from Mll-Af4/MIG (n = 12) generated from 3 independent donor mice were harvested at 160 to 200 days posttransplant. (F) Polymerase chain reaction detection of immunoglobulin rearrangement in genomic DNA from Mll-Af4/N-Ras^G12D preleukemic cells and in the spleen and bone marrow of primary leukemias. ***P < .001, *P < .05.