猪原始生殖嵴细胞(PGCs)建系因素的研究

Impact Factors on Establishment Cells Lines of Porcine Embryonic Germ

从五指山猪(WSZP)近交系第8～13代培育群中,先后选用21头5～10月龄青年母猪,分别于授精后25～30d采集胎儿106个,进行原始生殖嵴(PGCs)细胞分离、培养等建系技术研究。以DMEM+F10(1∶1)为基础培养液,按添加或不添加生长因子,将培养液分为A、B、C3种,并以STO细胞作饲养层,在38℃、5.0%CO2和湿润的气相中进行培养建系。结果获得胚胎生殖嵴细胞(EG)细胞系6个细胞株,其中1个EG细胞株传至11代、2个传至5代、1个传至4代、2个传至3代冻存。并进行了AKP染色、体外分化、冷冻-解冻复苏和嵌合体制作等鉴定研究。研究发现:不同胚龄对EG细胞建系具有一定影响,不同培养液对EG细胞建系效果不同,STO细胞饲养层的质量是建株、传代、冷冻-解冻复苏的关键因素之一。EG细胞系的初步建立,为今后筛选进入种系的EG细胞系、实施体外基因操作提供了可能。

The total 106 fetuses of Wuzhishan miniature pig (WZSP) were collected from 21 donors at day 25 to 30 of pregnancy (estrus=0 day), which was used in vitro culture, clone ,isolation, passage of porcine embryonic germ cells (PGCs). PGCs were collected from the genital ridge of the fetuses which were cultured on STO feeder cells in PEG's medium A (No. fetuses 24), B(No. fetuses 36), C(No. fetuses 46). The cultures were maintained at 38℃ in 5% CO_2air with media changed daily. 6 PEG like lines were deeply frozen and passaged F_3,F_4,F_5 and F_(11) respectively, which 3 of them were collected from day 27 of pregnancy only. The evaluation testing such as AKP staining and in vitro differentiation and passaging of frozen-thawing as well as making chimera pigs, have been carried out also. The results show that it could be effected on establishment of PEG cells line by embryos on different stages and different culture medium as well as the quality of STO.Under our experimental condition, PEG's will be survival in vitro after frozen-thawing, which is possible for future to select a fine PEG's line to make chimera and transgenic pig or definition in vitro.