cell type: Gr1/Mac1 tissue: Bone marrow strain: C57BL/6J location: bred and maintained in the animal research center of the British Columbia Cancer Agency, Vancouver, BC, Canada
Treatment protocol
Bone marrow cells were harvested from mice treated with 150 mg of 5-fluorouracil/kg 4 days before harvest and stimulated for 48 hours in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum (FBS), 10 ng/mL of human interleukin-6 (hIL-6), 6 ng/mL of murine interleukin-3 (mIL-3), and 20 ng/mL of murine stem cell factor (mSCF; all from StemCell Technologies Inc., Vancouver, Canada). MN1 was introduced into bone marrow cells by cocultivation with irradiated (4,000 cGy) GP+ E86 viral producer cells in the presence of 5 µg/mL protamine sulfate (Sigma-Aldrich, Oakville, Canada). Cells were then sorted for GFP expression and maintained in 6 ng/ml mIL-3, 10 ng/ml hIL-6, and 20 ng/ml mSCF. The transduced bone marrow cells from C57BL/6J mice were then injected into the tail vein of lethally irradiated syngeneic recipient mice that were exposed to a single dose of 750 cGy total-body irradiation accompanied by a life-sparing dose of 1 x 105 freshly isolated bone marrow cells from syngeneic mice. Donor chimerism was monitored by tail vein bleeds and FACS analysis of GFP expressing cells every four weeks. We have showed at the single cell level that CMPs, but not GMPs, are susceptible to MN1-induced transformation. To identify transcriptional differences between CMPs and GMPs that may explain this difference in susceptibilities to MN1 transformation we produced gene expression profiles of bone marrow cells from MN1 leukemic mice and mature myeloid bone marrow cells (Gr1+/CD11b+) from healthy mice and compared those to already published gene expression profiles of CMPs and GMPs (Krivtsov, A.V., et al. (2006). Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9. Nature 442, 818-822).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using Trizol reagent (Invitrogen), and RNA quality was assessed using the Bioanalyzer 2100 (Agilent Technologies, Mississauga, ON).
Label
Biotin
Label protocol
The labeling was done according to the manufacturer instructions.
Hybridization protocol
Hybridization was done on the Affymetrix GeneChip Mouse430 2.0 microarray according to manufacturer’s instructions. Protocol EukGE-WS2v5 Wash A1 Recovery Mixes 0 Wash A1 Temperature (C) 30 Number of Wash A1 Cycles 10 Mixes per Wash A1 Cycle 2 Wash B Recovery Mixes 0 Wash B Temperature (C) 50 Number of Wash B Cycles 6 Mixes per Wash B Cycle 15 Stain Temperature (C) 35 First Stain Time (seconds) 300 Wash A2 Recovery Mixes 0 Wash A2 Temperature (C) 30 Number of Wash A2 Cycles 10 Mixes per Wash A2 Cycle 4 Second Stain Time (seconds) 300 Third Stain Time (seconds) 300 Wash A3 Recovery Mixes 0 Wash A3 Temperature (C) 35 Number of Wash A3 Cycles 15 Mixes per Wash A3 Cycle 4 Holding Temperature (C) 25
Scan protocol
Pixel Size 1.56 Filter 570 Scan Temperature Scanner ID 50206370 Number of Scans 1 Scanner Type M10
Description
All arrays were performed at British Columbia Genome Sciences Centre, Vancouver, BC, Canada.
Data processing
Data were processed using MAS5 with default setting with GCOS 1.3.0.
