Mice were housed under controlled pathogen-free conditions (Harvard University’s Biological Research Infrastructure) and experiments were approved by the Harvard University Committee on the Use of Animals in Research and Teaching. Primary cortical progenitors were isolated from E14.5 brains and grown as neurospheres in presence of growth factors. Neurospheres were expanded for a maximum of one passages and then plated as a monolayer on dishes coated with poly-D-lysine (VWR) and laminin (BD). E14.5 pregnant dams were deeply anesthetized and the embryos were removed. Presumptive somatosensory area was microdissected in cold Hanks’ medium and mechanically triturated to obtain a single cell suspension. Cells were spun at 5,000 g for 5 min at room temperature and the pellet was resuspended in Neurosphere Media, containing 5,000U Penicillin/streptomycin, 200mM L-glutamine, N2 and B27 supplements (Gibco), as well as 20 ng/ml epidermal growth factor (EGF) (Sigma), and 20 ng/ml β-fibroblast growth factor (FGF) (Millipore) in order to promote the survival of neural stem cells. After quantification of cell density, neural stem cells were plated in 200 ml tissue culture flasks (untreated or low-adherence) (Corning) at 200,000 cells/flask and incubated at 37°C with 5% CO2. The medium was changed every 3–4 days and the cells were expanded as neurospheres for one passage. Subsequently, cells were plated as a monolayer on dishes previously coated with poly-D-lysine (VWR) and laminin (BD).
3XFLAG-Fezf2GFP and 3XFLAGGFP constructs were first cloned into pRETROX-IRES-ZsGreen1 retroviral vectors (Clontech Laboratories, Inc.) to generate high titer, replication-incompetent, VSVg-coated lentiviral particles that were packaged in 293T cells (MOI < 109 PFU/ml). 12-18 hours after plating, neural stem cells were infected with retroviruses. 16–20 h after infection, cells were switched into fresh media. Cells were collected 48 hours after infection.