All murine studies were performed with approval of the Animal Care and Use Committees of Northwestern University and Jesse Brown VA Medical Center. Mice with disruption of the IRF8 gene (Icsbp−/− mice) were obtained from Dr. Keiko Ozato (NIH, Bethesda, MD) [27 (link)].
Mononuclear cells were isolated from femurs of wild type or Icsbp−/− C57/BL6 mice and cultured in DME supplemented with 10% FCS, 1% pen-strep, 10 ng/ml GM-CSF, 10 ng/ml IL-3, 100 ng/ml Scf (R&D Systems Inc., Minneapolis MN). Lin-ckit+CD34+ cells were isolated from these cultures by magnetic bead affinity technique (Miltenyi Biotechnology, Auburn, CA) (referred to as “myeloid progenitor conditions”). Lin-ckit+CD34+ cells (with increased βcatenin expression) represent the LSC population in murine CP-CML [27 (link)]. Some cells were differentiated with 10 ng/ml G-CSF for 24 hrs (R&D Systems Inc., Minneapolis MN) (> 70% of cells are Gr1+ under these conditions; not shown).
Bcr-abl retrovirus and shRNA expressing lentiviruses were prepared by transfecting 293T cells with the relevant plasmid and the Ecopack plasmid [14 (link)]. Viral supernatants collected 48 hours post-transfection were titered in NIH3T3 cells. Murine bone marrow cells were transduced by incubation with viral supernatant (~107 pfu/ml) supplemented with polybrene (6 μg/ml) [14 (link)]. Transgene expression was confirmed by PCR and/or %GFP+ cells.
Mononuclear cells were isolated from femurs of wild type or Icsbp−/− C57/BL6 mice and cultured in DME supplemented with 10% FCS, 1% pen-strep, 10 ng/ml GM-CSF, 10 ng/ml IL-3, 100 ng/ml Scf (R&D Systems Inc., Minneapolis MN). Lin-ckit+CD34+ cells were isolated from these cultures by magnetic bead affinity technique (Miltenyi Biotechnology, Auburn, CA) (referred to as “myeloid progenitor conditions”). Lin-ckit+CD34+ cells (with increased βcatenin expression) represent the LSC population in murine CP-CML [27 (link)]. Some cells were differentiated with 10 ng/ml G-CSF for 24 hrs (R&D Systems Inc., Minneapolis MN) (> 70% of cells are Gr1+ under these conditions; not shown).
Bcr-abl retrovirus and shRNA expressing lentiviruses were prepared by transfecting 293T cells with the relevant plasmid and the Ecopack plasmid [14 (link)]. Viral supernatants collected 48 hours post-transfection were titered in NIH3T3 cells. Murine bone marrow cells were transduced by incubation with viral supernatant (~107 pfu/ml) supplemented with polybrene (6 μg/ml) [14 (link)]. Transgene expression was confirmed by PCR and/or %GFP+ cells.
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