Cell culture

The HEK293T (American Type Culture Collection (ATCC), #3216), Phoenix-ECO (ATCC #CRL-3214) and mouse 3T3 (ATCC #CRL-1658) fibroblasts cell lines were cultured in DMEM with 10% (v/v) FBS, 100 U ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin, 1 × GlutaMAX supplement (Gibco), 1 mM sodium pyruvate (Gibco) and 50 μm β-mercaptoethanol. These cells were maintained at 37 °C and 5% CO₂ in a humidified incubator. ES cells were cultured at 37 °C and 8% CO₂ in a humidified incubator. The medium for ES cells used was a 1:1 combination of Advanced DMEM/F-12 and Neurobasal medium, with 1 × N2 Supplement, 1 × B27 Supplement, 1 × L-Glutamine, 100 μm β-mercaptoethanol, 10³ U ml⁻¹ LIF, 3 μM CHIR99021 and 1 μM MEK inhibitor PD0325901. The ES cells used here were derived from an Sv129 ES cell line and have been previously described²⁷.