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通过电穿孔的方法,将含有新霉素抗性(Neor)基因和增强绿色荧光蛋白(EGFP)基因的双标记选择载体导入胎牛输卵管上皮细胞,获得了转基因细胞株.以转基因细胞为核供体,进行了牛的体细胞核移植.重构胚胎424枚,其中208枚体外发育至囊胚,囊胚发育率为49.1％.选择第7天转基因囊胚17枚移入17头同期发情的受体牛子宫角内,共有5头受体牛妊娠,妊娠率为29.4％.经过正常的体内发育,有3头转基因克隆牛出生,产犊率为17.6％.PCR和Southern检测结果表明,3头转基因克隆牛的基因组中都整合有外源目标基因.并且,绿色荧光蛋白在转基因克隆牛的耳部皮肤组织以及从耳部皮肤组织分离出的成纤维细胞中均有表达.上述结果显示,通过体细胞核移植技术可以有效生产转基因牛;所构建的双标记选择载体可以有效筛选出转基因细胞和转基因克隆胚胎,应用于转基因克隆动物的生产,确保所培育的克隆动物均为转基因动物.
The double marker selection vector containing Neor gene and enhanced green fluorescent protein (EGFP) gene was introduced into fetal calf epithelial cells by electroporation, and the transgenic cell lines were obtained. The body of a cow was subjected to somatic cell nuclear transfer.A total of 424 reconstructed embryos were reconstructed, 208 of which developed into blastocysts in vitro with a blastocyst rate of 49.1% .The 17 transgenic blastocysts selected on the 7th day were transferred into 17 estrus- In the uterine horn, a total of 5 recipient cattle were pregnant with a pregnancy rate of 29.4% .After normal in vivo development, 3 transgenic cloned calves were born with a calving rate of 17.6% .PCR and Southern test results showed that 3 transgenic In the cloned cattle, the foreign gene was integrated into the genome of the cow, and the green fluorescent protein was expressed in both the ear skin tissue of the transgenic cow and the fibroblast isolated from the ear skin tissue. The cell nuclear transfer technique can effectively produce transgenic cattle. The constructed double-marker selection vector can effectively screen the transgenic cells and the transgenic cloned embryos, and can be applied to the production of the transgenic cloned animals to ensure that Cloned animals are fertile transgenic animals.