DF1 chicken fibroblast cells (ATCC) were cultured at 39°C in DMEM with high glucose (ATCC) supplemented with 10% FBS, 2 mM l-glutamine, and 1% antibiotic-antimycotic. The DF1s were transfected with various RCAS plasmids using X-TremeGENE 9 (Roche). Primary brainstem gliomagenesis was induced by injecting NestinTVA mice (P3–5) with 1 × 105 virus-producing DF1 cells resuspended in 1 μL. A Hamilton syringe was used to inject the DF1 cells into the brainstem as previously described (34). DF1 cells expressing RCAS-PDGFB, RCAS-Cre, and RCAS-luciferase were injected in equal ratios for all studies except for the FACS experiments. In the FACS experiments, mice were injected with 1 × 105 virus-producing DF1 cells expressing RCAS-PDGFB, RCAS-Cre, RCAS-luciferase, and RCAS-GFP at a 2:2:1:1 ratio.

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