Leukemic transformation of Tg LT-HSCs conditionally expressing MLL-ENL. (A) Construction of the plasmid for conditional expression of MLL-ENL. CAG, hybrid CMV enhancer/chicken β-actin promoter; FLAG, tag sequence fused to the C terminus; SV40, simian virus 40. Arrowheads indicate loxP sites; filled diamond indicates triple stop codon sequence; horizontal arrows indicate primers to detect recombination between the loxP sites in (D); vertical arrow indicates the EcoR I site. (B) Expression of GFP in peripheral white blood cells from a conditional MLL-ENL Tg mouse (black line) compared with a WT littermate (gray shadow) on FACS. (C) Experimental strategy for myeloid immortalization assays and BMT. Sorted BM cells were retrovirally transduced with transgenes (dotted diagonal lines). (D) Recombination detected by genomic PCR in myeloid immortalization assays of WT and Tg LT-HSCs retrovirally transduced with mock (harvested at the end of the first plating) or CreER (Tg only; harvested at the end of each round of plating). G, germline configuration; R, recombination between loxP sites. (E) Expression levels of Hoxa9, Meis1, Evi1, and MLL-ENL by QRT-PCR in myeloid immortalization assays. Tg LT-HSCs (CD34[⁻]), Tg ST-HSCs (CD34[⁺]), Tg MPs, WT whole KSL cells, and WT MPs were retrovirally transduced with mock, CreER, or MLL-ENL and harvested at the first replating. (F) Myeloid immortalization assays of cells with retroviral transduction, as described in (E), by replating 10⁴ cells. (G-H) Typical morphology of the colonies of the Tg LT-HSCs retrovirally transduced with CreER at the end of the third round (G), and the immortalized cells constituting the colonies (H). Colonies, and cells stained with Wright-Giemsa, were viewed with an Olympus CKX41 microscope using a 4×/0.13 objective lens and an Olympus BX41 microscope using a 20×/0.5 objective lens, respectively. Images were acquired with Olympus DP21 and Olympus DP21 software. Magnification and bars in (G) indicate 40× and 200 μm; magnification and bars in (H) indicate 200× and 20 μm. (I) Immunophenotype of the immortalized Tg LT-HSCs (CD34^–KSL) and the WT whole KSL cells immortalized by retroviral transduction of MLL-ENL. (J) Survival curves of mice transplanted with Tg LT-HSCs (CD34^–; n = 4), ST-HSCs (CD34⁺; n = 4), whole KSL cells (n = 5 for CreER; n = 3 for mock), and MPs (n = 4), retrovirally transduced with CreER(-IRES-EGFP) or mock. The bar graphs indicate the mean ± SD of 3 independent experiments.