鸡胚生殖嵴中原始生殖细胞的分离培养

Isolation and culture of primordial germ cells from genital ridge in chicken embryos

目的 在鸡胚孵化的 19期以Ficoll密度梯度离心和酶解离两种方法分离生殖嵴中的原始生殖细胞 (PGCs)。探索在生殖嵴中PGCs分离、培养的适宜方式 ,以获得较多数量与较高活力的PGCs作介导生产转基因鸡。方法 在倒置显微镜下进行形态观察 ,台盼蓝染色比较存活时间 ,PAS特异染色法识别鉴定PGCs。结果 两种分离方法均能分离到一定数量的PGCs细胞。与Ficoll密度梯度离心法相比 ,酶解离法分离到PGCs的相对数量较多 ,存活时间较长 ,是一种较可行的分离方法。在鸡胚孵化的第 19期 ,PGCs大量聚集在肢体后端的生殖嵴原基处 ,此时的生殖嵴大小已达一定程度 ,分离其中的PGCs操作简便 ,有较强的可操作性。结论 提取的PGCs为转基因鸡的生产提供了介导材料。

Objective Along with two methods of ficoll density gradient centrifugation and EDTA trypsin collection of isolating primordial germ cells(PGCs)from chicken embryos from gential ridge at stage 19.Methods PGCs were then cultured in vitro in TCM 199 medium containing 10% FCS.The number of viable cells was determined by using the trypan blue test;PGCs were identified using PAS reaction.Results The results showed tha EDTA Trypsin collection was more preferable to PGCs collection.It′s the prime time for collection of PGCs from genital ridge in chicken embryos at stage 19.Conclusion Isolated PGCs were suitable for production transgenic chickens.