【作者】 王克振；

【导师】 周天鸿；

【作者基本信息】 暨南大学 ， 遗传学， 2009， 硕士

【摘要】 基因打靶技术已经越来越受到研究工作者的青睐,该技术也逐渐走向成熟。但是,传统的基因打靶所依赖的同源重组仅仅是个10^-6-10^-5之间的低概率事件。近几年以来,本课题组已经建立起了以基因组中度重复序列rDNA的间隔序列作为靶位点的多位点基因打靶技术,成功得到转基因鳜鱼和重组奶牛细胞,将打靶效率提高了200-300倍。但是,该技术离高效打靶还差很远。依据近几年的研究进展,国内外有很多卓有成效的基因打靶技术应用于动植物体内转基因。其中,最有效的就是锌指核酸酶（Zinc Finger Nuclease,即ZFN）的应用,它将基因定点整合率大大提高。本课题将锌指核酸酶的应用与多位点打靶相结合,理论上可将打靶效率提高到18%以上。本文经过筛选将人基因组上的中度重复序列rDNA的18S和5.8S之间的间隔序列ITS1作为靶位点,设计针对靶标的一对三联体锌指蛋白。分别与FokⅠ切割结构域连接,合成了一对锌指嵌合核酸酶。接着,将该对锌指核酸酶分别插入到真核表达核载体pcDNA3.1-NLS中,构建了一对真核表达载体pcS-ZFNs。利用PCR方法从pEGFP-N1中扩增pCMV-EGFP,从人基因组rDNA基因家族中靶基因插入位点两侧分别扩增两条基因打靶同源重组引导序列DS1和DS2,将DS1、DS2片段插入pMD19-pCMV-EGFP中,构建成功多位点基因打靶载体pMD19-DS1-pCMV-EGFP-DS2。本课题选取HEK293细胞作为研究对象,通过共转染一对pcS-ZFNs和基因打靶载体,得到了生长增殖稳定、绿色荧光持续表达的基因整合细胞。经提取基因组,进行靶位点验证性PCR试验,证实了基因的定点整合,并将定点整合效率提高到了20%以上。本课题的研究避免了药物的毒副作用,不经药物筛选就明显提高了基因打靶效率,为动物定点转基因技术的建立和人类遗传疾病的基因治疗提供了技术方法。更多还原

【Abstract】 The researchers have been more and more caring about the technique of gene target which is gradually coming to be mature.However,the traditional gene target is merely relied on homologous recombination,whose efficiency is just between 10^-6 and 10^-5.Recently,we have managed to establish a technique of multiple locus on gene targeting using the middle repeat of the interval sequence of rDNA as a target,and succeeded to produce the transgenic mandarin fishes and the recombination cow fertilized egg cell.Although in these researches the efficiency of gene target has been up to 200-300 folds,it is still a long way to real high efficiency. According to recent research,so many techniques about gene target with high efficiency have been put into use at home and abroad,in which a famous method is the application of zinc finger nuclease that can high improved the efficiency of gene target.Our research has combined the application of zinc finger nuclease with the multiple loci gene target,which can improved the efficiency of gene target up to 18%in theory.This article has used the ITS1 between 18S and 5.8S of rDNA as a target,and got a pair of three-zinc finger gene cutting the target sequence which combined with the coding sequence of Fok I slicing domain,thus we got a pair of zinc finger nucleases gene.And then,we inserted each zinc finger nuclease gene into the eukaryotic expression vector of pcDNA3.1-NLS,and got a pair of pcS-ZFNs.It was inserted into the pMD19-T vector for the construction of the recombinant vector pMD19-DS1-pCMV-EGFP-DS2 that the pCMV-EGFP gene which amplified by PCR using pEGFP-N1 out of MCS as template and two homogenous recombination arms of DS1 and DS2 which were amplified from human genome by PCR.By cotransfecting a pair of pcS-ZFNs and recombinant vector into HEK293 cells,we got the gene targeting cells with stable growth and reproduction,and persistent expression of the green fluorescence.After extracting the genome DNA of former HEK293 cells and testing the gene target by PCR,we managed to test the recombination efficiency of the gene target which is up to 20%.This research can avoid the bad reaction of the drug,improve the efficiency of gene target without drug selection and provide a transgenic technique for the transgenic animal and the gene therapy and diagnostics of human.更多还原