通用型基因打靶载体的构建及其功能鉴定  

Construction and Functional Analysis of a Universal Gene Targeting Vector

作　　者：刘晓蕾[1] 刘莹莹[1] 刘烨[1] 王毅[1] 王小海[1] 郭泽坤[1] 

机构地区：[1]西北农林科技大学生命科学学院,杨凌712100

出　　处：《农业生物技术学报》2014年第4期508-519,共12页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(No.31172279)

摘　　要：利用DNA同源重组(基因打靶)技术对动物基因组进行遗传修饰是转基因研究的重要手段之一。本研究以pEGFP-C1载体为载体骨架,成功构建了包含两个多克隆位点、两个同向LoxP(locus of crossing-over(x)in P1)序列和正负筛选标记基因的通用型打靶载体pGT-C1。正筛选标记新霉素磷酸转移酶基因(Neo)和水母绿色荧光蛋白基因(AcGFP)在两个LoxP序列之间;负筛选标记单纯疱疹病毒胸苷激酶基因HSV-tk和两个多克隆位点序列分别在两个LoxP序列外侧。经双酶切和测序证明载体构建成功后,转染胎牛成纤维(fetal bovine fibroblast,FBF)细胞,利用遗传霉素(G418)和丙氧鸟苷(GANC)分别对正负筛选标记进行功能验证;随后将Cre重组酶表达载体转染阳性FBF细胞,用半定量PCR检测Cre-LoxP系统的切割效率;最后构建针对牛(Bos taurus)β酪蛋白质基因的打靶载体pGT-L-R,并在转染药筛后对其打靶功能进行检测。打靶载体pGT-C1具有以下优点:包含两个多克隆位点,方便长短同源臂的定向插入,极大提高了该载体的通用性;位于两个同向LoxP之间的绿色荧光标记基因AcGFP可实时监控载体的转染效率和整合效率,还能依据荧光表达筛选去除筛选标记的阳性细胞,降低筛选标记在中靶细胞中可能产生的负面影响,为高效基因打靶提供了保证。综上所述,本研究成功构建了更加实用、有效的通用型打靶载体pGT-C1,以及针对牛β酪蛋白质基因的打靶载体pGT-L-R,并成功获得了转基因牛成纤维细胞单克隆,为转基因动物模型的建立提供了基础工具。Homologous recombination （gene targeting） is an important technique that is used to modify mammalian genome. We constructed an efficient common gene targeting vector pGT-C1 based on the plasmid pEGFP-C1. The vector consisted two different multiple cloning sites（MCS）, two locus of X-over P1 （LoxP） and positive and negative selection markers. Two positive selection markers, neomycin resistance gene （Neo） and aequorea coerulescens green fluorescent protein gene （AcGFP） flanked by two LoxP sites. The two synthesized multiple cloning sequences MCS1 and MCS2 were placed in outside of LoxP sites. Additionally, a negative selection marker HSV-tk （herpes simplex virus thymidine kinase） gene was located adjacent to MCS2. Each element was detected by enzyme digestion, and positive and negative selection markers were verified by adding geneticin（G418） or ganciclovir （GANC） to cell culture media, respectively. The cutting efficiency of Cre-LoxP system was measured by semi-quantitative PCR after transfected Cre expression vector. Finally, we built the cow （Bos taurus） beta casein targeting vector pGT-L-R, and tested the targeting efficiency after transfection and drug screening. Targeting vector pGT-C1 had the following unique features： it contains two multiple cloning sites （MCS）, which facilitated the directional insertion of homologous arms and then enhanced the versatility of this vector. The green fluorescent tag AcGFP between two LoxP was used to monitor instantly the transfection efficiency and integration efficiency, and the selection markers was able to be removed from the recipient cells by Cre-LoxP system to avoid the potential damage of selection markers to the recipient cells and increase the efficiency of gene knockout. Collectively, we constructed a gene targeting vector pGT-C1 with high efficiency and flexibility, and subsequently produced the transgenic bovine fibroblasts via constructing the vector pGT-L-R target the β-casein gene. This work provides basic tool for t

关 键 词：同源重组 通用型打靶载体 CRE-LOXP 转基因动物 

分 类 号：S823.2[农业科学—畜牧学]