扩增人肝细胞再生增强因子(human augmenter of liver regeneration, ALR)基因, 利用质粒pIRES2-EGFP构建新霉素(Neo)、增强绿色荧光蛋白(enhanced green fluorescence protein, EGFP)双标记基因且EGFP和ALR基因为双顺反子的真核表达载体。LipofectAMINETM介导其转染体外培养的绵羊胎儿成纤维细胞(Sheep fetal fibroblast cells, sFFCs); 经G418筛选转基因细胞; 激光共聚焦显微镜挑选绿色荧光单克隆细胞。PCR、RT-PCR和免疫组织化学方法进一步检测ALR基因及其表达; 稳定表达外源基因的sFFCs作供体, 移入去核的绵羊卵母细胞中, 进行体细胞核移植。通过激光共聚焦显微镜和ALR抗体检测EGFP、ALR基因在胚胎水平上的表达，其结果表明：由IRES连接的EGFP和ALR基因可在绵羊胎儿成纤维细胞内同时表达，由此细胞核移植产生的转基因胚胎在发育的各阶段均可见绿色荧光; 囊胚中所有细胞表达EGFP基因; 发绿色荧光的胚胎中ALR基因同时存在。因此，由IRES连接标记基因和目的基因, 以标记基因指示目的基因的表达, 可简化检测目的基因的繁琐手段; 用筛选的转基因早期胚胎进行移植, 可提高制备转基因动物的效率。
Human ALR gene sequence was amplified by PCR from human total DNA and inserted into pIRES2-EGFP vector. The bicistronic eukaryotic expression vector, pIRES-EGFP/ALR, expressing EGFP, Neor and ALR genes was constructed. Sheep fetal fibroblast cells (sEFCs) were transfected with pIRES-EGFP/ALR by the induction of lipofectAMINETM. The positive cell clones were selected with medium containing G418 (800 µg/ml). The fluorescence of transgenic cells was examined with a confocal laser scanning microscope. The expression of ALR gene was tested by PCR, RT-PCR and immuno-histochemical staining. The transgenic cells were used as donors for nuclear transfer to enucleated ovine oocytes. Transgenic embryos were tested by confocal laser scanning microscope and immuno -histochemical staining. Results showed that the EGFP and ALR genes linked with IRES were coexpressed simultaneously in sFFCs; the blastocysts formed by nuclear transfer using tranfected donor cells are all transgenic blastocysts. EGFP, ALR and Neor gene were all expressed in the transgenic embryos. In conclusion that a method to construct the positive embryos before pre-implantation which stably express ALR gene by the indication of EGFP expression has been successfully established. The application of this method can simplify the procedure of testing the targets and contribute to the efficiency increasing of transgenic domestic animal production.