鸡胚盘内转染外源基因的研究  

Transfect of Exogenous in Vivo of Chickens Germinal Disc

作　　者：李碧春[1] 孙怀昌[1] 陈国宏[1] 何光余[1] 肖小君[1] 吴圣龙[1] 冀得君[1] 

机构地区：[1]扬州大学动物科学与技术学院,扬州225009

出　　处：《畜牧兽医学报》2004年第5期481-486,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金项目(30170678)资助

摘　　要：采用显微注射法,分别将人生长激素(hGH)基因和人组织激肽释放酶基因(KLK1)直接注射到孵化24h的鸡胚中,继续孵化。在注射的320枚鸡胚中,孵化前7d死亡194枚,8～18d死亡108枚,孵出雏鸡18只,孵出率为5.6%(18/320)。8只雏鸡分别于出壳后的第1、2、3、5天死亡。7只于3月龄死亡。3只鸡(1公2母)存活至今,并开始产蛋。死亡雏鸡具有脚瘫、体弱、斜颈、站立不稳等异常表现,存活鸡无外观异常,但有产蛋紊乱现象。从孵化4d后死亡的150枚鸡胚和18只出壳雏鸡的组织提取基因组DNA,并用DNA杂交试验进行基因整合检测,结果发现其中的3枚鸡胚和3只雏鸡为人生长激素基因整合阳性,基因整合率为12.5%(6/48);18枚鸡胚和6枚雏鸡为KLK1整合阳性,整合率为22.2%(24/108)。结果表明:鸡胚盘内细胞通过显微注射法可以转染外源基因。To explore the possibility of generation of transgenic chickens by direct microinjection of transgenes into the germinal disc of developing embryos. 1.5 kb human growth hormone (hGH) mini gene fused with goat β-lactoglobulin (BLG) gene promoter and human tissue kallikein-1 mixed with LipofectAMine reagent, were injected respectively into the germinal disc of one-day incubated chicken embryos. Among 320 microinjected embryos,194 and 108 embryos died during the first seven days and 8～18 days post incubation ,respectively and 18 chickens were hatched with a hatch percentage of 5.6%(18/320). Both dot blotting and Southern blotting analyses showed that the BLG-hGH construct was integrated into the genomes of 3 out of 44 embryos died during 4 to 18 days post incubation and that of 3 hatched chickens. The KLK1 construct was integrated into the genomes of 18 out of 94 embryos died during 4 to 18 days post incubation and that of 6 hatched chickens. These data further demonstrated that direct microinjection of foreign genes into the germinal disc of developing embryos was a useful way of generation transgenic chickens.

关 键 词：鸡 胚盘 转基因 显微注射 基因整合率 

分 类 号：S831.3[农业科学—畜牧学]