Yuan Yuguo, Yu Baoli, Song Shaozheng, Zhou Feng, Zhang Liqing, Gu Yingying, Yu Minghui, Cheng Yong
Sheng Wu Gong Cheng Xue Bao. 2013 Nov;29(11):1573-80.
Gene knockout by ZFNs (zinc-finger nucleases) is efficient and specific, and successfully applied in more than 10 organisms. Currently, it is unclear whether this technology can be used for knocking-out enhanced green fluorescent protein (EGFP) gene in transgenic goats. Here we constructed and used ZFN-coding plasmids to produce genetic knockouts in the cells of cloned fetus produced from donor cells by microinjection of EGFP gene. Following introduced plasmids into caprine primary cultured fetus fibroblasts by electroporation, targeting of a transgene resulted in sequence mutation. Using the flow cytometric analysis, we confirmed the disappearance of EGFP expression in treated cells. Sequence from PCR products corresponding to targeted site showed that insertion of a G into the exon of EGFP resulted in frame shift mutation. These results suggest that ZFN-mediated gene targeting can apply to caprine fetus fibroblasts, which may open a unique avenue toward the creation of gene knockout goats combining with somatic cell nuclear transfer.
利用锌指核酸酶（ZFNs）进行基因敲除高效且特异，已成功应用于10多种生物。目前，尚不清楚该技术是否可用于敲除转基因山羊中的增强绿色荧光蛋白（EGFP）基因。在此，我们构建并使用了编码ZFN的质粒，以对通过显微注射EGFP基因从供体细胞产生的克隆胎儿细胞进行基因敲除。通过电穿孔将质粒导入山羊原代培养的胎儿成纤维细胞后，转基因的靶向作用导致了序列突变。利用流式细胞术分析，我们证实了处理过的细胞中EGFP表达消失。与靶向位点对应的PCR产物序列显示，EGFP外显子中插入一个G导致了移码突变。这些结果表明，ZFN介导的基因靶向可应用于山羊胎儿成纤维细胞，这可能为结合体细胞核移植创建基因敲除山羊开辟一条独特的途径。
