Doxycycline-inducible Dux ESC Line

Doxycycline (Dox)-inducible Dux ESC line was generated using the PiggyBac Transposon System in our laboratory (hereafter referred to as ESC^DUX), in which Dux expression was induced in the presence of Dox^{21 (link)}. The ESC^DUX was cultured in ESC medium (10% FBS, 1× sodium pyruvate, 1× GlutaMAX, 1× MEM non-essential amino acids solution, 1× penicillin/streptomycin and 0.055 mM β-mercaptoethanol in DMEM high glucose (4.5 g liter⁻¹)) containing 2i (1 µM PD0325901, LC Laboratories; and 3 µM CHIR99021, LC Laboratories) and LIF (1,000 U ml⁻¹, in-house) on cell-culture plates coated with 0.1% gelatin under feeder-free conditions. The expanded colonies were dissociated using 0.05% trypsin-EDTA solution for passaging.