鸡胚原始生殖细胞的分离和鸡鸭嵌和体的制备

Production of chicken/duck chimeras by isolating and transferring chicken circulating primordial germ cells into duck embryos

探索了鸡胚 12～ 17期血液中的原始生殖细胞 (PGCs)迁移数量变化规律 ,将其在液氮中冷冻保存。并以Ficoll密度梯度离心、MiniMACS磁气分离、滤膜三种方法对PGCs进行分离 ,结果发现 12～ 17期血液中均有PGCs存在 ,13期达到高峰约 4 7 1± 10 5个 /μl,在血液中比例为 0 0 12 6 %。冷冻保存 3个月后解冻成活率达 80 %以上。三种分离方法所得的分离效果分别为 95 7%、39 2 %、6 3 0 % ,纯度为2 7 5 %、8 4 %、3 1%。将分离的原始生殖细胞以微注射法转移至 14～ 15期麻鸭胚胎中制备了鸡鸭种间嵌和体 ,获得 8只雏鸭 (8/110 )。以鸡W染色体探针原位杂交法在早期鸭胚性腺中检测到鸡原始生殖细胞 ,嵌和率达 84 2 % (16 /19)。表明鸡原始生殖细胞能够迁移定居到鸭胚性腺中 ,并有可能增殖分化成有功能的配子。

Primordial germ cells(PGCs) are the progenitor of the gametes.Avian PGCs originate from the epiblast, circulated in the blood stream,migrated into the developing germinal ridge.In this research,blood was collected from stage 12～17 chicken embryos. The number of PGCs was counted under inversed microscope after PAS reaction. Concentration of primordial germ cells in bood reached peak in stage 13(47.1±10.5,0.0126%).Blood contained PGCs were stored in liquid nitrogen for 3 months. PGCs were isolated by Ficoll density gradient centrifugation, MiniMACS immunomagnetic purification and filter film.Respectively 95.7%,39.2%,63.0% PGCs in blood were isolated with the purity about 27.5%,8.4% and 3.1%. 100～200 isolated PGCs were microinjected into dorsal aorta of stage 14 maya duck. The injected embryos were incubated until hatched. eight putative chimera hatchlings were obtained. To identify chicken PGCs in gonad of injected duck embryo,in situ hybridzation was performed with chick W chromosome specific DNA probe.84.2%(16/19) gonad sections in different ages showed positive reaction at some extent. These results suggested that chick circulating PGCs could migrate and proliferate into duck gonad together with duck PGCs. Chicken/duck germline chimeras could be produced by transfer cirulating PGCs.