Murine MLL-AF9 Leukemia Model

Mouse MLL-AF9 leukemic cells with Dot1l^fl/fl or wild-type genotypes were generated by transformation of mouse bone marrow Lin⁻Sca1⁺cKit⁺ (LSK) cells with retrovirus expressing MLL-AF9 fusion protein and transplanted into sublethally irradiated recipient mice as described previously^{8 (link)}. The leukemic blasts harvested from the diseased mice were cultured in vitro in IMDM plus 15% FBS supplemented with 20 ng/ml murine SCF (PeproTech), 10 ng/ml murine IL-3 (PeproTech) and 10 ng/ml murine IL-6 (PeproTech). Human leukemic cell lines Molm-13, MV4-11 and HL-60 were maintained in RPMI plus 10% FBS. All cell culture medium contained L-Glutamine (2mM; Gibco), penicillin (100 units/ml; Gibco), streptomycin (100 ug/ml; Gibco) and plasmocin (5 ug/ml; InvivoGen). Human cell lines including HL-60, MV4-11 and Molm-13 were tested in March - May, 2013 for authentication by short tandem repeat (STR) profiling performed by ATCC. Live cell counts were obtained by high-throughput flow cytometry with SYTOX-blue cell death dye exclusion (Life Technologies) using an LSRFortessa-HTS Analyzer (BD biosciences).

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Chen C.W., Koche R.P., Sinha A.U., Deshpande A.J., Zhu N., Eng R., Doench J.G., Xu H., Chu S.H., Qi J., Wang X., Delaney C., Bernt K.M., Root D.E., Hahn W.C., Bradner J.E, & Armstrong S.A. (2015). DOT1L inhibits SIRT1-mediated epigenetic silencing to maintain leukemic gene expression in MLL-rearranged leukemia. Nature medicine, 21(4), 335-343.

Publication 2015

murine SCF murine IL-3 murine IL-6 L-Glutamine penicillin streptomycin plasmocin SYTOX-blue cell death dye exclusion LSRFortessa-HTS Analyzer

Cell Cell death Cell l Cell lines Cells bone marrow Cells culture Ckit Culture medium Flow cytometry Genotypes Glutamine Human Mll protein Murine Penicillin Plasmocin Retrovirus Sca1 Short tandem repeat Streptomycin Transplanted recipient

Corresponding Organization :

Other organizations : Memorial Sloan Kettering Cancer Center, Broad Institute, Massachusetts Institute of Technology, Dana-Farber Cancer Institute, Children's Hospital Colorado, University of Colorado Denver

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Protocol cited in 4 other protocols

Variable analysis

independent variables

Dot1l^fl/fl (genotype)

dependent variables

Leukemic cell growth and survival

control variables

Mouse bone marrow Lin^- Sca1^+ cKit^+ (LSK) cells
Retrovirus expressing MLL-AF9 fusion protein
Culture media (IMDM + 15% FBS, supplemented with murine SCF, IL-3, and IL-6)
Penicillin, streptomycin, and plasmocin in culture media
Human leukemic cell lines (HL-60, MV4-11, Molm-13)

controls

Positive control: Wild-type genotype
Negative control: Not mentioned

Annotations

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