【作者】 李建许；

【导师】 刘红林；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2004， 硕士

【摘要】 鸡胚盘细胞在第X期还未分化，所以在引入同期发育的受体胚胎时能够参与受体各种组织的发育。本试验以狼山鸡为供体，从肉鸡为受体，通过赤道开窗法制作了狼山鸡-AA肉鸡的嵌合体。试验共操作了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，嵌合率为20.4％，其中15只孵化成活，成活个体中嵌合体比例为78.9％。对2只嵌合体公鸡解剖发现，有一只的睾丸发育异常，通过PCR扩增禽类W染色体特异性的重复序列发现，两只嵌合体公鸡的性腺都嵌合了供体异性的细胞。这一结果表明我们所采用的嵌合体制作方法对于下一步转基因鸡的制备是可行的。 通过对狼山鸡、AA肉鸡和嵌合体鸡的12种行为观察发现，在站立、蹲卧、站立修饰羽毛、站立啄环境、走动或跑动等行为上，狼山鸡的发生频率比AA肉鸡高，其差异极显著(P＜0.01)。比较发现，嵌合体1的这些行为的发生频率都位于狼山鸡相应行为平均值的95％的置信区间之內，其行为模式倾向于供体狼山鸡，嵌合体2和6分别在洗浴和站立行为的发生频率上与供体狼山鸡相似，而其他3只嵌合体鸡的行为模式却与受体AA肉鸡接近。这一制作嵌合体的方法还可用于禽类神经行为学的研究。 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小时开始有外源基因的表达，体外培养12小时后获得了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天后在8个鸡胚中检测到有外源基因的存在，阳性率为40％(8/20)。更多还原

【Abstract】 The X stage avian blastodermal cells have not yet differentiated, and when they were introduced into the same stage embryos of recipients. They could take part in the development of different tissues of the recipients. In the experiment, the langshan is used as the donor and the AA broiler is 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, and the chimera rate was 20.4%. 15 chimeras survived, and the survived chimera rate was 78.9%. Abnormal testises were 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.By observing the 10 behaviors of the langshans, the AA broilers and the chimeras, Significant differences (p<0.01) were found on standing, sitting, stand preening, stand environment pecking, walking or running between the two control breeds, and the frenquency on these behaviors of chimeral were all located in 95% confidence intervals for means of the langshan, so its behavioral patterm prefered for the donor langshan. Chimera2 and 4 were similar to the langshan chickens separately on the performance of dustbathing and standing, while the behavioral pattern of the other three chimeras were close to the recipient. The method of producing chimeras can also be used in avian neuroethology.Plasmid pEGFP was introduced into the X stage avian balstodermal cells via lipofectamine in vitro, the exogenous gene began to express. After cultured for 12h in vitro the transfecting rate reached 21.43%. The transfected blastodermal cells were injected into the subgerminal cavity of the recipient, after bing hatched for 6 days the exogenous genewere detected in 40% (8/20) of the embryos examined.更多还原