Neuronal Differentiation of Ehmt1 Mutant mESCs

Analysis used two independent clonal populations of mESC Ehmt1^+/− cell lines mutant mouse ES cells (mESCs), obtained from the European Mouse Mutant Cell Repository Centre (EuMMCR); each had a single copy of a ‘knockout first’ conditional allele Ehmt1^{tm1a(EUcOMM)Hmgu} allele [32 (link)]. Control (Ehmt^flp) a cell lines were generated using the flp-allele to restore a wild type gene. mESCs were grown on gelatin-coated plates in knockout DMEM (Gibco), supplemented with ESC certified FBS (Invitrogen), L-Glutamine (Gibco), 2-mercaptoethanol (Sigma) and ESGRO leukaemia inhibitory factor (LIF) (Chemicon) at 37 °C. Feeder-free neuronal differentiation was initiated in media lacking LIF for 4 days and 5 μM Retinoic acid was added to the culture until day 8. At this stage, cells were dissociated with 0.05% trypsin (Sigma) and seeded at 1.5 ×10⁵ per cm² density on Poly‐D-ornithine (Sigma)/laminin (Roch) in N2 medium (Sigma) [33 (link)].