Research Center; and of a deceased autologous transplant donor through the Bone Marrow Transplant Laboratory at Northwestern Memorial Hospital after Institutional Review Board approval. Cells were prestimulated and transduced with retrovirus as described. After 46 h, GFP positive cells were isolated using a MoFlo highspeed sorter and expression of the transfected gene was confirmed 
by immunoblotting with anti-HA tag antibody. Long-term liquid culture of primary cells was performed in the presence of a cytokine cocktail as previously described. Colony-forming cell and long-term cultureinitiating cell assays were performed as previously described. Cytospin preparations of cells harvested from the CFC plates were stained with Giemsa and a 500-cell differential count was performed using an Olympus BX51 microscope. Cells with blast and promyelocyte morphology were counted as primitive; those with myelocyte/metamyelocyte morphology as intermediate myeloid; those with band, segmented neutrophil, monocyte, and macrophage morphology as mature myeloid; those with intermediate hemoglobinization as intermediate erythroid; and those with full hemoglobinization as mature erythroid. Photomicrographs were taken with an Olympus DP71 camera with a 606oil objective. Materials and Methods Plasmid construction The pTracer-CMV/Bsd construct 22441874 expressing HA-tagged NUP98-HOXA9 was previously described. The N51S mutation was created in this construct by replacing a BglII/XbaI fragment with a synthetic fragment containing an A to G substitution in the 51st codon of the homeodomain. The mutation was confirmed by sequencing. MSCV-IRES-GFP constructs expressing HA-tagged NUP98-HOXA9, and HOXA9 have been described. NUP98-HOXA9/N51S and HOXA9DN were subcloned upstream of IRES into MSCV-IRES-GFP. For luciferase constructs, the following promoter regions were amplified from human genomic DNA by PCR using PfuUltra high-fidelity DNA polymerase: HOXB6 from 21934 to +81; JUN from 22013 to +89; KBTBD10 from 22369 to +76; PLN from 21937 to +81; and SERPINE1 from 22012 to +74. PCR products were subcloned into pGL4.11 upstream of the luciferase gene using the NheI/EcoRV sites for HOXB6, JUN, and KBTBD10 promoters; the KpnI/XhoI sites 17594192 for the PLN promoter; and the XhoI/ EcoRV sites for SERPINE1 promoter. Deletions of the KBTBD10 and PLN promoters are shown in Fig. 4A and 4B; they were similarly subcloned into pGL4.11. All sequences generated by PCR were confirmed by DNA sequencing. Flow cytometry Flow cytometry was performed on a FACScan flow 80321-63-7 site cytometer upgraded to 5 colors and two lasers, and analyzed using FCS3 Express and FlowJO v8.6.3 software. The antibodies used for these studies were CD11b from eBioscience; CD235a from BD; and CD33 and CD45 from Beckman Coulter. Microarray analysis of primary human CD34+ cells Sorted GFP-positive cells were snap-frozen 3 days after transduction and submitted to the Siteman Cancer Center Laboratory for Clinical Genomics where total RNA was isolated and target preparation and microarray hybridization were performed. Labeled targets were hybridized to Affymetrix HGU133 Plus 2.0 GeneChip microarrays. Data were merged with the Transformation by NUP98-HOXA9 updated gene annotation data for each probe set on the array using Spotfire DecisionSite 9.1.1 for Functional Genomics. Probe sets were filtered according to present/absent calls and the sets that were absent across all chips were filtered from the analysis. The fold change of each probe set
