Retrovirus transduction and generation of leukemia

The MLL-AF6 construct, consisting of 35-347 amino acids of the AF6 portion¹², was cloned into MSCV-puro. MLL-AF6 and MLL-AF9¹⁵ retroviruses were produced in BOSC23 cells. For transduction, FACS-sorted LSK cells were seeded in Retronectin (Takara)-coated dishes containing retroviral supernatants for 48 h and the transduced cells were expanded for 7 days in methylcellulose M3234 (Stem Cell Technologies) supplemented with cytokines (6 ng/mL IL-3, 10 ng/mL IL-6, and 20 ng/mL SCF). For MLL-AF6 AML, after puromycin selection (2 μg/mL) 2 × 10⁵ cells were injected into sublethally (650 rads) irradiated CD45.1 congenic mice to generate leukemia. For MLL-AF9 AML, 2 × 10⁵ cells after transduction were injected into sublethally irradiated congenic mice. For generation of secondary leukemia, 2 × 10⁵ primary leukemic cells were injected into sublethally (650 rads) irradiated congenic mice. PB was obtained every month after transplantation and analyzed by Hemavet HV950FS (Drew Scientific) and FACS. Cells were stained with anti-CD45.1 and CD45.2 antibodies to distinguish donor-derived cells from the host cells, as well as anti-CD11b and Gr1 antibodies to identify leukemia cells. A recipient mouse was considered positive if donor-derived cells were present and also constituted more than 0.3% of the cells in the PB. BM cells harvested from moribund mice were cytospun and stained with Giemsa’s azur-eosin-methylene blue (Merck Millipore).