Generation of MLL-AF9 and MLL-ENL knock-in genes by genome engineering. (A) Schematic illustration of experimental strategy to induce a DSB by TALENs followed by integration of the knock-in template in the MLL gene locus by homology-directed repair (HDR). (B) FACS profiles show fluorescence of K562 and CD34⁺ cells nucleofected with the knock-in template alone (gray line) or in combination with the MLL TALENs (black line) sorted on days 5 and 3, respectively. (C) Summary of NeonGreen expression in K562 (n[AF9] = 3, n [ENL2/7] = 2) and CD34⁺ cells (n[AF9] = 11, n[ENL2] = 7, n[ENL7] = 5) pre- and postsort. *P < .05 was considered statistically significant. Error bars indicate standard error of the mean (SEM). (D) Confocal microscopy images show NeonGreen expression in sorted K562 and control cells as indicated. Top row, cell density (brightfield); bottom row, NeonGreen expression detected by GFP excitation (450-490 nm) and ×10 objective. (E) PCR/RT-PCR was performed on gDNA and cDNA isolated from NeonGreen-positive K562 and CD34⁺ cells to detect integration and transcription of the construct under control of the endogenous MLL promoter (representative results shown for MLL-AF9). BGH, bovine growth hormone.