Mouse MLL-AF9 Leukemia Model

Mouse MLL-AF9 leukemic cells were generated by transformation of mouse BM Lin⁻ cells with retrovirus expressing MLL-AF9 fusion protein and transplanted into sublethally irradiated recipient mice as described previously (5 (link)). The leukemic blasts harvested from the diseased mice were cultured in vitro in Iscove's modified Dulbecco's medium (IMDM) (Gibco) plus 15% fetal bovine serum (FBS) supplemented with murine SCF (20 ng/ml; PeproTech), murine interleukin-3 (IL-3) (10 ng/ml; PeproTech), and murine IL-6 (10 ng/ml; PeproTech). Human cell lines MV4-11, MOLM13, KOPN-8, RS4;11, MonoMac6, NOMO1, THP-1, Daudi, CCRF-CEM, HL-60, Ramos, Jeko-1, Jurkat, and Kasumi-1 cells were maintained in RPMI (Gibco) supplemented with 10% FBS. Human cell lines NCI-H661, U251, and HepG2 were cultured in Dulbecco’s modified Eagle’s medium (DMEM)(Gibco) supplemented with 10% FBS. All cell culture medium contained l-glutamine (2 mM; Gibco), penicillin (100 U/ml; Gibco), streptomycin (100 μg/ml; Gibco), and plasmocin (0.5 μg/ml; InvivoGen). All cells were cultured in a 37°C incubator with 5% CO₂. Cells stably expressing the Cas9 endonuclease were established via transduction of lentiCas9-Blast (52962, Addgene) lentivirus and selected by blasticidin (Gibco).