Generation of MLL-AF9 and MLL-ENL Mouse Models

Flt3^ITD/+ mice26 (link) were kindly provided by Gary Gilliland and crossed with Rosa26^Cas9/+ mice6 (link). Freshly isolated bone marrow from 6- to 10-week-old female Rosa26^Cas9/+, Flt3^ITD/+; Rosa26^Cas9/+ or moribund Npm1^flox−cA/+; Flt3^ITD/+ mice was used. Bone marrow cells were exposed to erythrocyte lysis (BD PharmLyse, BD Bioscience), followed by magnetic bead selection of Lin^- cells using the Lineage Cell Depletion Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Lin^- cells were cultured in X-VIVO 20 (Lonza) supplemented with 5% BIT serum substitute (Stem Cell Technologies), 10ng ml^-1 IL3 (Peprotech), 10ng ml^-1 IL6 (Peprotech) and 50ng ml^-1 of SCF (Peprotech). Retrovirus constructs pMSCV-MLL-AF9-IRES-YFP and pMSCV-MLL-ENL-IRES-Neo were used with package plasmid psi-Eco to produce retrovirus. 293T cells (Life Technologies) were cultured and prepared for transduction in 10cm plates as described above. For virus production, 5 μg of the above plasmids and 5 μg psi-Eco packaging vector were transfected drop wise into the 293T cells using 47.5 μl TransIT LT1 (Mirus) and 600 μl Opti-MEM (Invitrogen). Transduction of primary mouse cells was performed in 6-well plates as mentioned above. After transduction, cells were sorted for YFP (for MLL-AF9) or selected with neomycin (for MLL-ENL).