利用ZFN敲除转基因猪细胞中多拷贝EGFP基因

Unsatisfied Removal of Multiple Exogenous EGFP Gene Inserts in Trangenic Porcine Somatic Cells by Zinc-finger Nucleases

锌指核酸酶（zinc finger nuclease,ZFN）是由特异性识别DNA的锌指结构域和Fok I切割结构域组成,能够在基因组特定位点上切割DNA,引起DNA双链断裂（double-strand break,DSB）.通过DSB修复机制,可以使基因修饰的效率比传统方法提高102~104倍.目前,利用ZFN对动物内源基因进行敲除的研究较多,但对转基因动物中外源多拷贝基因进行敲除的报道较少.本研究首先利用荧光定量PCR法对本实验室培育的两头转基因猪中增强型绿色荧光蛋白（enhanced green fluorescent protein,EGFP）基因的拷贝数进行鉴定,发现其拷贝数分别为11.95和17.36拷贝;然后将靶向EGFP的一对ZFN转染进拷贝数为17.36的EGFP转基因猪的成纤维细胞中,并通过流式和CEL-1酶切方法检测敲除效率.结果表明,转染400 ng、800 ng和1 200 ng ZFN的切割效率分别为0.97%、1.39%和1.76%,可见随着转染ZFN剂量的增加,ZFN的切割效率逐渐提高.但是,不发绿色荧光的细胞比例却没有明显提高,因此认为,ZFN敲除转基因动物中多拷贝基因的效率还是比较低.

Recombinant zinc finger nuclease（ZFN） is engineered with site-specific zinc finger domains and the cleavage domain of restriction enzyme Fok I. ZFNs have been showed to improve gene targeting efficiency by 100 to 10 000 folds by introducing DNA double-strand breaks in the target genes. Increasing studies employed ZFN to knockout endogenous gene. In two EGFP transgenic pigs of the study, the copy number of transgenic pig was estimated as 11.95 and 17.36, respectively by quantitative real-time PCR. EGFP-transgenie ZFN plasmid was constructed and introduced into the transgenic pig fibroblast through electroporation. Using survey or nuclease assay（Cel-1 assay） , we found that percentages of the cleavage of target sequence were 0.97% , 1.39% and 1.76% after transfecting 400, 800 and 1 200 ng of the ZFN plasmids. The gene knockout efficiency was detected by fluorescence cytometry analysis and assays to detect mutations. However, the flow cytometric analysis indicated that the percentage of non-fluorescentcells was not significantly improved after ZFN treatment. The results implied that ZFNs might works less efficiently to disrupting multicopy genes in transgenic animals.