Generation of transgenic hESCs expressing MLL-AF4 and/or FLT3-activating mutations. (A) Schematic representation of the lentiviral vectors used. (B) Phase-contrast morphology (top) and fluorescence microscopy (bottom) of representative hESC colonies from FLT3-TKD-, FLT3-ITD-, MLL-AF4-, MLL-AF4/FLT3-TKD-, and MLL-AF4/FLT3-ITD-hESCs. (C) RT-PCR confirming expression of the MLL-AF4 transcript in transduced hESCs. GAPDH was used as a housekeeping gene. (D) PCR confirming the presence of either FLT3-TKD or FLT3-ITD mutations in transgenic hESCs. Vectors containing the FLT3 mutations were used as positive controls. (E) Phosphosignaling analysis of transduced hESCs, showing increased AKT, ERK, and STAT5 phosphorylation (relative to EV-hESCs) in cells transduced with FLT3-activating mutations.