翻译为英文：22性腺中PGCs的分离与培养选取如皋黄鸡和寿光黑鸡的新鲜种蛋将其在37度孵化55天以获得Hamburger and Hamilton HH 27–28阶段的鸡胚。在体式显微镜下使用精细镊子单独分离每个胚胎的性腺。每个胚胎的性腺单独转移到300μl含有01 BSA的磷酸盐缓冲液盐水PBS中进行清洗离心后去除上清。将性腺组织在200ul 025胰蛋白酶-EDTA中孵育1分钟同时用移液器将

2.2 Isolation and culture of PGCs in gonads

Freshly laid eggs from Gaoyou yellow chickens and Shouguang black chickens were incubated at 37°C for 5.5 days to obtain chicken embryos at Hamburger and Hamilton (HH) stages 27-28. Using fine forceps under a dissecting microscope, the gonads of each embryo were isolated individually. Each gonad was washed in 300 μl of phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA), centrifuged and the supernatant was removed. The gonad tissue was incubated in 200 μl of 0.25% trypsin-EDTA for 1 minute and then mechanically dissociated into a suspension using a pipette. The digestion was stopped by adding 300 μl of DMEM culture medium containing 5% fetal bovine serum (FBS), and the suspension was centrifuged and the supernatant was removed. The cell pellet was resuspended in FAcS culture medium and transferred to a 48-well culture plate for cultivation. The medium was changed every 3 days by replacing 1/3 to 1/2 of the medium, until the cell number reached 1×10^6 for cryopreservation.

The chicken PGC culture medium was prepared according to Whyte et al. (2015). The FAcS culture medium was composed of DMEM (high glucose, no glutamine, no calcium) (Gibco, 21068028) as base medium, supplemented with H2O (Sigma, W3500), 0.15 mM CaCl2 (100x) (Sigma, C7902), 1x B-27® Supplement (50x) (Gibco, 17504044), 2.0 mM GlutaMAXTM-1 (1000x) (Gibco, 35050061), 1x MEM NEAA (100x) (Gibco, 11140050), 0.1 mM 2-mercaptoethanol (55 mM) (Gibco, 21985023), 0.20% chicken serum (Gibco, 16110082), 1x EmbryoMax® Nucleosides (100x) (Sigma, E8-008-D), 1.2 mM sodium pyruvate (100 mM) (Sigma, 11360070), 0.20% ovalbumin (Sigma, A5503), 0.01% sodium heparin (1000x) (MCE, HY-17567A), 1x FGF-2 (2500x) (MCE, K128N), and 25 ng/ml human activin A (MCE, HEK293)