鸡胚原始生殖细胞的分离和鸡鸭嵌合体的制备研究  

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

作　　者：刘春海[1] 李赞东[1] 黄劲松[1] 沙金[1] 危华[1] 赵晨[1] 孙明军[1] 

机构地区：[1]中国农业大学生物学院,北京100094

出　　处：《河北农业大学学报》2001年第3期62-66,共5页Journal of Hebei Agricultural University

基　　金：国家自然科学基金资助项目 (39780 0 2 8)

摘　　要：探索了 12～ 17期鸡胚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Fi coll密度梯度离心、MiniMACS磁气分离、滤膜 3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 (4 7 1± 10 5 )个·μL-1,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。3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合体 ,获得 8只雏鸭 (8/ 110 )。以鸡W特异性DNA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 ,嵌合率达 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 。Primordial germ cells (PGCs) are the progenitor of the gametes Avian PGCs originate from the epiblast, circulate in the blood stream, migrate 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 blood reached the peak in stage 13 (47 1±10 5, 0 012 6%) 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% and 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 embryos 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 hybridization was performed with chicken 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 germlin chimeras could be produced by transferring circulating PGCs

关 键 词：原始生殖细胞 嵌合体 分离 鸡 鸭 迁移规律 冷冻保存 

分 类 号：Q954.43[生物学—动物学]