Murine recombinant stem cell factor scf

Animal studies were performed according to the ACUC-approved protocols at Northwestern University and Jesse Brown VA.
Bone marrow mononuclear cells were obtained from femurs of C57/BL6 mice and cultured for 24 h in DME with 10% fetal calf serum, 1% penicillin–streptomycin, 20 ng/ml murine GM-CSF (R&D Systems Inc., Minneapolis, MN, USA), 20 ng/ml murine recombinant IL3 (R&D Systems) and 100 ng/ml murine recombinant stem cell factor (Scf; R&D Systems; 2 × 105 cells/ml). Cells were maintained 48 h in GM-CSF+IL3+Scf followed by isolation of CD34+ cells (using the Miltenyi magnetic bead system, Miltenyi Biotechnology, Auburn, CA, USA) or differentiation with 20 ng/ml of G-CSF.^{12 (link)}Retrovirus (~107 PFU/ml) was generated with HoxA10/MSCV, HoxA9/MSCV, E76K-Shp2/MSCV or control MSCV using the Phoenix packaging line according to manufacturer's instructions (Stratagene). Bone marrow mononuclear cells were cultured 24 h in 20 ng/ml IL3, 20 ng/ml GM-CSF and 100 ng/ml SCF, incubated with retroviral supernatant supplemented with polybrene (6 pg/ml) and cultured 48 h in GM-CSF, IL3 and Scf±G-CSF.^{12 (link)} Transduced cells were selected in the appropriate antibiotic (G418 or puromycin with the various vectors) post transduction. We find that 70–75% if the cells are consistently transduced (preselection) with this technique.