AML from Mll^PTD/WT:Flt3^ITD/WT mice have molecular features similar to those reported for human MLL-PTD⁺ and/or FLT3-ITD⁺ AML. (A) Quantitative real-time RT-PCR was performed using CD117⁺ BM cells sorted to > 95% purity from age-matched mice ≥ 50 weeks of age to measure the copy numbers of the mouse Mll-PTD and the Mll-WT transcripts. Results demonstrate reduced Mll-WT copy number only in leukemic Mll^PTD/WT:Flt3^ITD/WT mice. Figure is representative of 2 age-matched cohorts of mice. (B) Relative real-time RT-PCR measuring HoxA9 expression in whole BM cells from age-matched mice > 50 weeks of age. (C) Total Flt3 mRNA was measured by relative real-time RT-PCR using BM cells of age-matched mice ≥ 50 weeks of age. (D) Genomic PCR reactions using 150 ng DNA and genotyping primers to amplify both the Flt3-ITD and Flt3-WT loci were performed to demonstrate a reduction or loss of Flt3-WT in primary Mll^PTD/WT:Flt3^ITD/WT AML samples taken from moribund mice. Multiple samples from each animal are defined as follows: lane 1, tail DNA from genotyping at 4 weeks; lane 2, whole BM DNA from the time of death when moribund with AML (50-80 weeks); and lane 3, sorted leukemic Ly5.2 Mll^PTD/WT:Flt3^ITD/WT AML blasts from secondary transplant of Mll^PTD/WT:Flt3^ITD/WT AML blasts into Ly5.1 recipients. The Ly5.2 → Ly5.1 adoptive transfer was used to obtain a pure population of Mll^PTD/WT:Flt3^ITD/WT AML blasts for analysis. Five representative samples are shown. Reactions that did not produce any bands for Flt3-WT and Flt3-ITD were removed as noted by the black bar.