Retroviral transduction of c-Kit-enriched bone marrow (anti-CD117 microbeads; Miltenyi Biotec, Auburn, CA, USA) with NICD in the MSCV-IRES-GFP (MIG) retrovirus (8 (link)) was performed as previously described (17 (link)). Recipient mice were transplanted by retro-orbital injection following a split dose of 11.0 Gy of irradiation. For T-ALL rescue, Nr4a1 and Dnmt3a were cloned into MSCV-IRES-mCherry (MIC) to transduce primary GFP+ T-ALL cells. 4×104 GFP+mCherry+ cells were transplanted into secondary recipients. For secondary transplantations, leukemic cells were transplanted into sublethally irradiated (6.0 Gy) mice. The TtRMPVIR retroviral backbone (Addgene, Cambridge, MA, USA) was modified to place Nr4a1-2A-mCherry under the control of a tetracycline-responsive promoter. Sample size was calculated based on published studies for NICD transduction and transplantation (8 (link), 18 (link)) to provide at least 80% power to compare a median survival difference of 25% based on two-sided two-sample test for proportions (p<0.05). NICD transduction and transplantation was performed independently for primary mice five times.
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Notch1-Driven T-ALL Modeling
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Corresponding Organization : Washington University in St. Louis
Other organizations : University of Michigan–Ann Arbor
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Variable analysis
independent variables
- Retroviral transduction of c-Kit-enriched bone marrow with NICD in the MSCV-IRES-GFP (MIG) retrovirus
- Transduction of primary GFP+ T-ALL cells with Nr4a1 and Dnmt3a in MSCV-IRES-mCherry (MIC)
- Modification of the TtRMPVIR retroviral backbone to place Nr4a1-2A-mCherry under the control of a tetracycline-responsive promoter
- Survival outcome of recipient mice transplanted with transduced cells
- Recipient mice were transplanted by retro-orbital injection following a split dose of 11.0 Gy of irradiation
- For secondary transplantations, leukemic cells were transplanted into sublethally irradiated (6.0 Gy) mice
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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