Avian PGC Expansion and Gene Editing

PGC lines were derived from individual fertile eggs, cultured in FAOT medium and expanded to 400,000 cells in 5 weeks before performing gene editing experiments^{38 (link)}. Fertile eggs bred from Hy-line layer lines were incubated for 2.5 days and then 1 μl of embryonic blood was taken from the dorsal aorta of HH stage 16 HH embryos and placed into FAOT medium. FAOT medium contains custom-made Avian Knockout DMEM (Life Technologies #041-96570 M) 1× B-27 supplement (Life Technologies #17504044), 2.0 mM GlutaMax (Life Technologies #35050-038), 1× non-essential amino acids (Life Technologies #11140050), 1× EmbryoMax nucleosides (Merck Millipore #ES-008-D), 0.1 mM β-mercaptoethanol (Life Technologies #31350010), 0.2% ovalbumin (Sigma-Aldrich A5503), 1.2 mM sodium pyruvate (Life Technologies #11360070), 0.15 mM CaCl2, 0.01% sodium heparin, 4 ng/ml h-FGF2 (R&D Systems), 50 ng/ml ovotransferrin (Sigma-Aldrich C7786) and 25 ng/ml activin A (Peprotech). PGCs were grown at 37 °C in a 5% CO₂ atmosphere and fed every 48 h.