通用型基因打靶载体的构建及其功能鉴定

Construction of Universal Vector for Gene Targeting and Analysis of Its Function

摘要:

为了构建适合大多数基因座位点打靶的通用型基因打靶载体及打靶成功后去除正选择标记基因, 以克隆载体pGEM-3Z为骨架, 插入了一个正选择标记基因新霉素磷酸转移酶基因(neo)、两个相同的负选择标记基因单纯疱疹病毒胸苷激酶基因HSV-tk1和HSV-tk2, 并在neo的两侧各添加了一个方向相同的LoxP(locus of crossing-over (x) in P1)序列及两个不同的多克隆位点序列, 从而构建了载体pA2T。插入的两个不同的多克隆位点序列中, neo和HSV-tk1之间的多克隆位点序列有8个稀少的酶切位点、neo和HSV-tk2之间的多克隆位点序列有5个稀少的酶切位点, neo、HSV-tk1和HSV-tk2有各自独立的转录单元。脂质体法转染山羊成纤维细胞, 用遗传霉素(G418)和丙氧鸟苷(GAC)进行正负筛选, 验证了正负选择标记基因的生物活性, 证明通用型基因打靶载体pA2T构建成功。载体pA2T转化组成性表达Cre重组酶(Cyclization recombination protein)的大肠杆菌BM25.8, 检测到LoxP序列的生物活性, 结果表明pA2T中的正选基因可以被Cre重组酶去除。因此, 本研究所构建的通用型基因打靶载体pA2T, 根据不同的基因座设计同源臂后, 插入到MCS中可直接用于不同基因座位点的打靶, 并能够在打靶成功后用Cre重组酶去除基因组中插入的neo基因, 为用基因打靶的方法制作转基因动物提供了便利。

Abstract:

To make a universal gene targeting vector fitting for most gene and delete positive selection gene after targeting successfully, a vector named pA2T was constructed by inserting one neomycin gene (neo) for positive selection and two same herpes simplex virus thymidine kinase gene HSV-tk1 and HSV-tk2 for negative selection into the vector of pGEM-3Z, and two locus of crossing-over (x) in P1 (LoxP) and two different multiple cloning sites (MCS) were inserted into two flanks of neo separately. There were eight rare cloning sites between neo and HSV-tk1 and five rare cloning sites between neo and HSV-tk2, and neo, HSV-tk1 and HSV-tk2 could be translated respectively in the pA2T. Transfection of the pA2T into goat fetus fibroblast cells with LipofectamineTM 2000 conferred resistance to geneticin (G418) and resistance to ganciclovir (GAC) in the cells, which suggested the positive and negative selectable markers could express in the cells and thus the vector pA2T could be used as a universal gene targeting vector. Transformation of the pA2T into the BM25.8 expressing Cre recombinase conferred neo was deleted in the pA2T, which suggested the LoxP was active. Thus, this vector can be inserted by most gene sequences as homologous sequences and positive selection gene can be deleted after targeting successfully, which is very convenience for the production of transgenic animals using gene targeting method.