Derivation and Culture of Avian Primordial Germ Cells

PGC line derivation and culture were carried out as described (Whyte et al., 2015 (link)). Briefly, 1 μl blood isolated from a stage 16 HH embryo (Hamburger and Hamilton, 1992 (link)) was placed in culture medium containing 1× B-27 supplement (Thermo Fisher Scientific), 0.15 mM CaCl₂, 2.0 mM GlutaMax (Thermo Fisher Scientific), 1× non-essential amino acids (Thermo Fisher Scientific), 0.1 mM β-mercaptoethanol, 1× EmbryoMax nucleosides (Merck Millipore), 1.2 mM pyruvate (Thermo Fisher Scientific), 0.2% ovalbumin (Sigma) and 0.01% sodium heparin (Sigma). 25 ng/ml activin A (Peprotech), 4 ng/ml FGF2 (R&D Systems) and 5 µg/ml ovotransferrin (Sigma) were added to Avian Knockout DMEM (osmolality: 250 mOsmol/kg, 12.0 mM glucose, calcium chloride free; Thermo Fisher Scientific, a custom modification of Knockout DMEM). Chicken serum at 0.2% (Biosera) was added to this medium to produce FAOTcs medium. A male and a female PGC line were derived in FAOTcs medium and expanded to 2.5×10⁵ cells in 5 weeks before use in targeting experiments.