鸡Ⅹ期胚盘细胞体外培养

CULTURE OF CHICKEN STAGE X BLASTODERM CELLS

为证实经遗传修饰的鸡X期胚盘细胞具有参与受体胚胎发育和形成嵌合体的能力 ,本研究将由鸡X期胚盘制成的细胞悬液与经脂质体包埋的抗鸡传染性支气管炎病毒基因重组质粒PGS1共孵育后 ,直接显微注入同期受体胚盘 (14 0枚 ) ;或对转染后供体细胞进行G418抗性筛选后显微注入同期受体鸡胚盘 (14 0枚 ) ;或将供体细胞体外培养 4 8h ,再与脂质体 PGS1复合物共孵育后显微注入同期受体鸡胚盘 (190枚 ) ,制备转基因嵌合体鸡 ,并应用PCR和RAPD方法 ,对鸡胚和雏鸡不同组织或血液中的DNA进行检测。结果表明 :直接注射组孵化率(5 7% )显著 (P <0 0 1)高于G418筛选处理组 (1 4 % )和培养 4 8h处理组 (2 1% ) ;G418筛选处理组不同胚龄鸡胚组织、器官中外源DNA的PCR检测阳性率均高于其它二个组。实验结果证明 ,体外培养 4 8h并经遗传修饰的胚盘细胞仍然具有形成嵌合体的能力 ,利用早期胚盘细胞途径制备转基因鸡是可行的。

Blastoderm cells modified genetically from stage X embryos of chicken could still enter recipient embryos at the same stage of development and were the able to produce chimeras. Donor cells suspended from stage X blastoderms and recombinant plasmid P GS1 containing the infectious bronchitis virus S1 gene were embedded in liposome and co cultured. The co culture was then treated in three ways: 1. Microinjected into the receptors ( n =140)at the same stage of development directly. 2. The co culture was anti screened with G418 before being microinjected. 3. In order to produce chimeras donor cells were cultured in a monolayer in vitro for 48 h then co cultured with recombinant plasmid P GS1 before being microinjected into the receptors( n =190). PCR and RAPD analyses of DNA from the blood of young chickens and embryonic tissue indicated that the hatchability of the first group(5 3%) was significantly higher than that of the second(1 4%) and the third (2 1%) (One Way ANOVA, P <0 05). The positive rate of exogenous DNA amplification in embryonic organs from the second group was higher than that from the first or third group. These results indicate that donor cells cultured in vitro for 48 h and co cultured with plasmid DNA might migrate into recipient embryonic tissue to produce chimeras. It is, therefore, potentially possible to produce transgenic chickens using lipofection mediated exogenous gene transfer to blastoderm cells isolated from stage X embryos. Subsequent injection of these cells into receptors at the same stage of development could result in the production of germline chimeras.