狼山鸡-AA肉鸡嵌合体的制作及其转基因鸡研究

Production of Langshan-AA chimeras and transgenic study via chimeras

试验Ⅰ:以狼山鸡为供体,AA肉鸡为受体,通过赤道开窗法制作了狼山鸡-AA肉鸡的嵌合体。共操作了108枚鸡蛋,孵化成活19只,孵化成活率17.6%。获得表型嵌合体鸡胚22枚,其中15只孵化成活,嵌合体率为13.9%。对2只嵌合体公鸡解剖发现,有1只的睾丸发育异常。PCR扩增禽类W染色体特异性的重复序列发现,2只嵌合体公鸡的性腺都嵌合了供体异性的细胞。结果表明所采用的嵌合体制作方法制备转基因鸡是可行的。试验Ⅱ:利用脂质体介导法,在体外将外源pEGFP质粒转入到鸡X期胚胎细胞中,培养8 h开始有外源基因的表达,体外培养12 h后获得了21.43%的转染效率。将转染后的鸡胚盘细胞移入到受体AA肉鸡的胚下腔,孵化6 d后在8个鸡胚中检测到有外源基因的存在,阳性率为40%(8/20)。

Experiment Ⅰ： The Langshan was used as the donor and the AA broiler was the recipient, the Langshan-AA chimeras were produced via windowing the recipient eggs. 108 eggs were manipulated, and 19 chickens were hatched. The hatching rate was 17. 6%. 22 chimeric embryos were obtained, 15 chimeras survived. The survived chimera rate was 13.9%. Abnormal testises was founded in one of the two male chimeras by dissecting. Hetero-sexual cells were found in the gonads of both chimeras by PCR amplifying the W-specific repeating sequences. The result showed that it was possible to produce transgenic avians by using our method of producing chimeras. Experiment Ⅱ ： Plasmid pEGFP was introduced into the X stage chicken balstodermal cells via lipofectamine in vitro, the exogenous gene began to express 8 h later. After cultured for 12 h in vitro the transfecting rate reached 21.43%. The transfected blastodermal cells were injected into the subgerminal cavity of the recipient, after being hatched for 6 days the exogenous gene were detected in 40% （8/20） of the embryos examined.