Experimental design of HOXB4 target gene analysis in transplanted murine HSCs. (A) Schematic diagram of HOXB4 retroviral and GFP control vectors used to transduce 5-fluorouracil (5-FU)–treated BM cells. (B) Timeline of transduction and mouse transplantation assay. C57Bl/6J mice were treated with 5-FU for 3 days prior to isolation of BM cells. After 2 days of prestimulation with a cytokine cocktail, cells were transduced for 4 days with either the GFP or HOXB4 retroviral vector. A total of 1 to 2 million cells were transplanted into lethally irradiated recipient C57Bl/6J mice. Vector-expressing LSK cells were isolated from the BM at 6, 12, and 20 weeks posttransplantation and were processed by flow sorting prior to total RNA isolation. (C) Flow cytometry sorting strategy for transduced LSKs. BM cells isolated from recipients were first gated for lineage-negative cells, then for cKit⁺ Sca-1⁺ marker status, and then for vector expression based on GFP or yellow fluorescent protein (YFP) expression. Sorted GFP/YFP⁺ LSK cells were used for subsequent gene expression analyses. BMC, BM cells; BMT, BM transplant.