Wehi 3b

Bone marrow cells were harvested from 6 week old B6.129S4-Il2rg^tm1Wjl/J mice (Jackson Laboratory, Bar Harbor, Maine). Cells were subjected to red cell lysis in NH₄Cl solution (0.8% NH₄Cl with 0.1 mM EDTA). Lineage-cells were enriched using a Lineage Cell Depletion Kit according to the manufacturer’s protocol (Miltenyi Biotech, Germany). Cells were pre-stimulated at 37°C in DMEM with 15% FCS, 15% WEHI-3B, murine IL-3 (7 ng/mL), IL-6 (12 ng/mL), and SCF (56 ng/mL) (Stem Cell Technologies, Canada). JAK3 mutants and IL2Rγ retroviruses were generated as described above. After 24 hrs, the cells were spinoculated into 6-well plates with viral supernatant in pre-stimulation medium in the presence of 5 μg/mL polybrene and HEPES at 2,500 rpm for 90 mins. This was repeated at 48 hrs. Cells expressing JAK3 and IL2Rγ were doubly sorted by FACS, gated on GFP (JAK3) and anti-human IL2Rγ-PE (BioLegend). 1×10⁴ cells per 35 mm dish were plated in triplicate in methylcellulose medium in the absence of cytokines (MethoCult M3234, Stem Cell Technologies). Colonies were scored following 8 days of incubation at 37°C in 5% CO₂.