To generate AML on an Erg-deficient background, c-Kit-enriched BM from Ergfl/fl and Ergfl/fl; R26-CreER mice were transduced with MLL-ENL-IRES-GFP (Ohlsson et al. 2014 (link)) and transformed through three passages in colony assays (Stem Cell Technologies, M3231) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 20 ng/mL murine SCF (mSCF) (PeproTech), 10 ng/mL human IL-6 (hIL-6) (PeproTech), 10 ng/mL human GM-CSF, and 10 ng/mL murine IL-3 (PeproTech). Ergfl/fl and Ergfl/fl; R26-CreER transformed colonies were subsequently dissociated and passaged in RPMI 1640 (Invitrogen) supplemented with 20% FCS, 20% WEHI-conditioned medium, 20 ng/mL mSCF, and 10 ng/mL hIL-6 ± 0.5 μM 4-OHT to allow for excision of Erg. Finally, serial replating assays were performed as described in Ohlsson et al. (2014) (link). For T-ALL analysis, c-Kit-enriched BM from Ergfl/fl and Ergfl/fl; Cd2iCre mice was transduced with NOTCH-ICD (Chiang et al. 2008 (link)) or NrasG12D in which luciferase was replaced with Venus (Zuber et al. 2009 (link)) and transplanted to irradiated (900 cGy) Ly5.1 recipients (CD45.1). Log rank test was used to analyze differences in survival.
Protocol detail
AML Generation in Erg-deficient Model
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Assays
C kit
Conditioned medium
Gm csf
Human
Ires
Luciferase
Mice
Penicillin
Stem cell
Streptomycin
T all
Corresponding Organization :
Other organizations : University of Copenhagen, Rigshospitalet, Lund University, Skåne University Hospital
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Variable analysis
independent variables
- Erg deficiency
- Presence or absence of Erg excision (using 4-OHT)
- Transformation of c-Kit-enriched BM cells
- Ability of transformed cells to undergo serial replating
- Development of T-ALL
- Transduction of c-Kit-enriched BM cells with MLL-ENL-IRES-GFP
- Culture conditions for transformed cells (RPMI 1640 with 20% FCS, 20% WEHI-conditioned medium, 20 ng/mL mSCF, and 10 ng/mL hIL-6)
- Transduction of c-Kit-enriched BM cells with NOTCH-ICD or NrasG12D-Venus
- Irradiation of recipient mice (900 cGy)
- None specified
- Erg^fl/fl mice (without Cre-mediated Erg excision)
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