鸡胚胎干细胞无饲养层培养体系的建立及其转染体系的优化

Establishment of Feeder-free Culture System of Chicken Embryonic Stem Cells and Exploration of an Optimized Transfection System

鸡胚胎干细胞(cES)以其全能性,在研究胚胎发育生物学和家禽育种等方面有巨大的应用前景。本实验旨在建立cES无饲养层培养体系和对LipofactiminTMLTX(脂质体)介导转染体系进行优化。采用药匙法从新鲜受精蛋中分离获取cES,接种于层粘连蛋白包被的培养皿,待形成大克隆后,用口吸管剥离法吸取cES克隆进行消化传代,并采用生化和免疫学方法鉴定其干细胞活性。设计质粒量800、900、1 000 ng 3个水平,质粒、脂质体比为1∶1.5、1∶2、1∶2.5体系转染cES。结果表明:无饲养层培养体系体外培养cES细胞能稳定传代至第6代,鉴定结果显示碱性磷酸酶阳性和阶段特异性胚胎抗原1阳性,指明克隆能保持未分化状态;质粒900 ng、质脂比为1∶2时,可获得最佳转染效率72%,为cES的脂质体介导外源基因的转染提供参考。

Chicken embryonic stem cells is a valuable model for genetic modification, developmental biology research and the potentialities of regenerative therapies. Obviously, feeder-layer culture system is cumbersome for screening after transfection. The cES cells were isolated from blastoderm of the stage X embryos of chicken, and seeded on laminin coated culture until the formation of large clones, passaged cES clones by mouth sucker method, detected alkaline phosphatase activity and the corresponding antigen expression. Several groups, plasmid 800, 900, 10 00 ng, lipofaetmin LTX 1.5, 2, 2.5 times of plasmid, were used to transfect cES cells. The result showed that cES cells can stably passaged to 6th generation culture in vitro using feeder-free culture system, expressing strong alkaline phosphatase （AKP） activity and stage-specific embryonic antigen-1 indicating the maintenance in the state of undifferentiated. Using lipofactminTM LTX mediated transfection by 900ng plate, can obtain a high transfection efficiency about 72%. These provide an plasmid and 18μL liposome on 12-well alternative tool for genetic modification ofchickensand lay a solid foundation for the application of cES cells.