作　　者： ; ; ; ; ; ; ; ; ; ; ; ;

机构地区： 佛山大学医学院

出　　处： 《科学通报》 2012年第9期711-719,共9页

摘　　要： 该研究旨在建立锌指核酸酶介导的多位点基因打靶技术,为获得稳定遗传的转基因动物或基因治疗临床应用解决技术难题.首先利用OPEN平台设计、构建能识别人基因组内rDNA基因间隔序列的锌指蛋白基因序列,与FokⅠ的切割结构域连接、表达后获得锌指核酸酶基因,再构建锌指核酸酶真核表达载体.另外,构建含有2条同源重组引导序列和绿色荧光蛋白基因(EGFP)的多位点基因打靶载体.将锌指核酸酶真核表达载体和多位点基因打靶载体共转染HEK293细胞,内参对照PCR-灰度分析法检测外源基因定点整合效率,结果显示单独转染多位点基因打靶载体的定点整合效率为6.8%;而由于锌指核酸酶在染色质DNA上切割rDNA基因的间隔序列,诱导高效同源重组,锌指核酸酶载体、多位点基因打靶载体共转染的定点整合效率为24.2%,较常规基因打靶定点整合率(10-6～10-5)提高了24000多倍.共转染的HEK293细胞在无任何筛选的条件下持续培养2个月,经过20次传代之后,子代细胞能够持续表达EGFP,提示表达稳定.本研究建立了锌指核酸酶介导的高效多位点基因打靶技术,不仅大大提高了外源基因的定点整合效率,而且兼顾了基因表达的稳定性和安全性,为动物定点转基因和人类基因治疗提供了重要的技术平台,具有广泛的应用前景. To address the technical problems of producing stable transgenic animals and clinical applications of gene therapy, the present study aimed to establish a multi-locus gene targeting technique mediated by zinc finger nucleases （ZFN） that enables efficient site-specific integration and stable expression of foreign genes. First, the OPEN method （oligomerized pool engineering） was used to design four pairs of genes of zinc finger proteins （ZFP） that identify the internal transcribed spacer （ITS） of human ribosomal DNA （rDNA） genes. To obtain ZFN genes, the ZFP genes were synthesized by PCR and connected to the DNA sequences of the cutting domain of the endonuclease, Fok I. These ZFN genes were then cloned into eukaryotic expression vectors. In addition, vectors for multi-locus gene targeting containing two homologous recombination direct sequences and the EGFP gene were constructed. The two types of vector were co-transfected into HEK293 cells and efficient homologous recombination was induced by the ZFN cleaving the target sites of ITS of the rDNA genes. Site-specific integration of foreign genes was detected by internal reference control PCR and the gray analysis method. The efficiency using only multi-locus gene targeting vectors was 6.8%, while it was 24.2% following co-transfection of both the eukaryotic ZFN expression vectors and the multi-locus gene targeting vectors. Compared to the efficiency of 10-6-10-5 for conventional gene targeting, the efficiency of site-specific integration of foreign genes has been greatly improved （increased by more than 24000 times）. After co-transfected HEK293 cells were cultured for two months （20 generations） without any drug selection, cells continued to express GFP. This study indicates that the technique of multi-locus gene targeting mediated by ZFN can not only greatly improve the efficiency of gene targeting, but can also produce stable expression of transgenes. A new technology platform for producing site-specific transgenic animals and with sig

关 键 词： 锌指核酸酶 基因打靶 多位点 同源重组 定点整合 稳定表达 转基因动物 基因治疗

领　　域： [生物学]