Setbp1 cooperates with BCR/ABL to transform committed myeloid progenitors. (A) Survival curves of lethally irradiated C57BL/6-Ly5.2 mice receiving 1 × 106 in vitro expanded BM progenitors infected with MSCV-BCR/ABL-IRES-GFP virus, pMYs-Setbp1-IRES-GFP virus, or the combination. Results are combined from 3 independent experiments with each containing the 3 indicated groups. Transduction efficiencies: 28%-42% for BCR/ABL groups; 19%-25% for Setbp1 groups; and 21%-31% for cotransduction groups. Secondary recipients receiving 1 × 106 spleen cells from primary leukemic mice died of leukemia between 11 and 13 days as indicated by the gray bar (n = 5). (B) A typical enlarged leukemic spleen (right) compared with a control normal spleen (left). (C) H&E staining of a typical liver section from leukemic mice displaying liver infiltration by the leukemic cells. Original magnification ×100. (D) Wright-Giemsa staining of cytospin preparation from the BM of a leukemic mouse. Original magnification ×400. (E) Staining of GFP positive leukemic cells from the BM and spleen (SP) of a typical moribund mouse with indicated antibodies. Numbers represent the percentages of gated events. (F) Semiquantitative reverse transcription PCR analysis of total RNA extracted from normal mouse bone marrow (NBM), leukemic BM, and spleen (SP) from 3 leukemic mice (L1, 2, and 3) using indicated gene-specific primers. β-actin was included to control for RNA loading. (G) Southern blotting analysis of genomic DNA from leukemic spleens (L1 to L7) using a GFP-specific probe. Samples were digested by EcoRI, and each band represents a separate integration. (H) Survival curves of lethally irradiated C57BL/6-Ly5.2 primary recipient mice receiving purified mouse GMPs (1-1.5 × 105 cells/recipient) infected with MSCV-BCR/ABL-IRES-GFP virus, pMYs-Setbp1-IRES-GFP virus, or the combination as well as secondary (2nd), tertiary (3rd), and quaternary (4th) recipients of the cotransduction group receiving 1 × 106 spleen cells from preceding leukemic mice. Transduction efficiencies: 50%-60% for BCR/ABL groups; 40%-45% for Setbp1 groups; and 34%-50% for cotransduction groups.
