5:1 under neurosphere culture conditions. Transduced cells were selected with blasticidin for 7 days. Primary transduced neurospheres were dissociated into single

cells and replated at 10 cells/μl in 6-well plates for secondary neurosphere formation assays. For each construct, tertiary neurosphere assays were performed to confirm the results seen in secondary neurosphere assay. The expression of each construct was confirmed with western blot and immunocytochemistry. For total cell population analyses, total neurospheres from each 6-well were collected and mechanically dissociated into single cells. Total viable cells were counted with hemocytometer using 0.2% trypan blue exclusion. Total neurosphere numbers were counted under a dissection microscope. The images of individual neurospheres were taken using a 4× objective, and ImageJ software selleck chemicals llc was used for measuring neurosphere size (area). Neurospheres isolated from E14.5 Olig2-null embryos were stably transduced with retrovirus expressing eGFP, Olig2-wt-V5, or Olig2-TPN-V5. Transduced neurospheres were

dissociated into single-cell suspension, and 50,000–150,000 cells were injected into the lateral ventricles of homozygous newborn Shi mice (Jackson Laboratory). Recipient mice were sacrificed at postnatal day 30, perfused, and postfixed overnight with 4% paraformaldehyde. Brains were dissected, sectioned at 50 μm, and stained for chicken anti-GFP (Aves Labs), rat anti-MBP (Chemicon), and rabbit

anti-V5 (Invitrogen). Cells were labeled buy HA-1077 for 2 hr in phosphate-free media supplemented with 300 μCi/ml 32P orthophosphate. Cells were washed in cold PBS and protein extracts generated using RIPA buffer. Olig2 was immune precipitated using a pan-olig2 antibody (Arnett et al., 2004) and resolved using SDS-PAGE. Quantitation of the signal was performed using a Typhoon Trio (GE Healthcare). Phosphorylation state-specific antibodies were before generated and purified as described previously (Alberta and Segal, 2001) using a peptide containing the triple phosphorylation motif (LVSpSRPpSpSPEPDDLC) conjugated to KLH using the Imject Conjugation kit (Pierce) as antigen in rabbits by Covance (Denver, PA, USA). Two Ink4A−/−ARF−/− Olig2−/− EGFRVIII neurosphere lines (EB5 and EB7) were generated as described ( Ligon et al., 2007), and then stably transduced with eGFP, Olig2 WT, and Olig2 mutants of interest via retroviral infection and five passages of neurosphere expansion. For each Olig2 construct, serial dilutions of cells at 1 × 105, 1 × 104, 1 × 103, and 1 × 102 were injected into the right striatum of Icr-SCID mice (Taconic Farms, Inc.) at the coordinates: A, −0.5 mm; L, 1.50 mm; and D, 2.65 mm, relative to the bregma. Animals were sacrificed at the onset of neurological/clinical symptoms.