摘要
本研究采集孵化 14期鸡胚血液中的原始生殖细胞 ,在液氮中冷冻保存三个月后 ,解冻成活率达 80 %以上。以Ficoll密度梯度离心将其从血液中分离纯化 ,分离获得纯度为 2 7 5 % ,平均每枚胚胎可获得近 5 0个PGCs。将分离的鸡PGCs10 0～ 2 0 0个以微注射法转移至 15期早期麻鸭胚胎中制备了鸡鸭种间嵌合体 ,孵出 8(6♂ ,2♀ )只雏鸭 ,总孵化率为 7 3% (8/110 )。以鸡W染色体特异性DNA探针原位杂交法在早期鸭胚性腺中检测鸡PGCs ,在被检测的 2 1个鸭胚的性腺中 ,16个有不同程度的阳性信号 ,嵌合率达 76 2 % (16 /2 1)。实验结果表明鸡原始生殖细胞能够迁移并定居到鸭胚性腺中 ,并有可能在鸭性腺中增殖分化成有功能的配子。
Blood samples were collected from stage 14 chicken embryos, dispersed in MEM medium containing 10% FBS and 10% DMSO, frozen to -80℃ in a BICELL freezing vessel, then stored in liquid nitrogen for three months. The survival rate of the thawed PGCs was confirmed by the Typan Blue exclusive method. Over 80% of thawed PGCs were viable. PGCs were isolated from the thawed and fresh blood of chicken embryos by Ficoll density gradient centrifugation. The concentration of PGCs at stage 14 was 0 0126%, and the concentrated fraction contained PGCs at about 27 5% purity. About 50 PGCs could be obtained from each embryo. After PGCs were thawed and isolated, about 100～200 PGCs in one microliter were injected into the dorsal aorta of each stage 15 Maya duck embryo from which one microliter blood had been drawn prior to injection for the purpose of reducing recipient PGCs. The recipient embryos were incubated until hatching. Of 110 Maya duck embryos injected with chicken PGCs, 7 3%(8/110) survived to hatching. Duck PGCs were also isolated from the recipient embryo blood. About 30 PGCs could be obtained from each duck embryo. To identify the presence of chicken PGCs in the gonads of recipient duck embryos, in situ hybridization (ISH) was performed with a chicken W chromosome specific DNA probe labeled with digoxigenin. The specificity of the chicken W chromosome repetitive DNA probe was determined by hybridization of the probe with female chicken blood and duck blood. Female chicken blood had a positive reaction while duck blood had negative results in all three samples. Seventy six percent of duck gonads (16/21) at different ages of development showed positive reaction at various levels after ISH. These results suggest chicken PGCs, could migrate, colonize and proliferate in duck gonads, together with duck PGCs, during development. Transferring PGCs could produce chicken/duck germline chimeras.
出处
《动物学报》
SCIE
CAS
CSCD
北大核心
2002年第4期543-548,共6页
ACTA ZOOLOGICA SINICA
基金
国家自然科学基金 (No .3 9780 0 2 8)~~
关键词
鸡
鸭
异种间嵌合体
制备
原始生殖细胞
Chicken, Duck, Primordial germ cells (PGCs), Inter species chimeras
