Culture of zebra finch PGCs

Gonads retrieved from zebra finch embryos at HH 28 were enzymatically digested and then cultured in vitro using a previously reported method²¹. Briefly, gonadal cells were suspended in PGC culture medium, which comprised knockout Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 2% chicken serum (Millipore Sigma, Burlington, MA, USA), 1 × nucleosides (Millipore Sigma), 2 mM l-glutamine (Thermo Fisher Scientific), 1 × nonessential amino acids, β-mercaptoethanol, 10 mM sodium pyruvate, 1 × antibiotic–antimycotic reagents (Thermo Fisher Scientific), and 10 ng/mL human basic fibroblast growth factor (Millipore Sigma). The PGCs-enriched suspension was sub-cultured every 4–5 days for about 1 month, and a fraction of adherent cells was used as a feeder layer.