A one-step cloning method for the construction of somatic cell gene targeting vectors: application to production of human knockout cell lines.
A one-step cloning method for the construction of somatic cell gene targeting vectors: application to production of human knockout cell lines.
复制标题
一种构建体细胞基因打靶载体的一步克隆方法：在人敲除细胞系生产中的应用
DOI：
10.1186/1472-6750-12-71
复制
发表时间：
2012-10-09
影响因子：
3.5
通讯作者：
Du R
中科院分区：
文献类型：
--
作者：
Liu Y;Li S;Zhang H;Wan Z;Zhang X;Du R
背景基因打靶技术是研究基因功能的有效手段。传统上，敲除（KO）载体的构建需要扩增步骤以获得基因组DNA的两个同源的大片段。限制性内切酶，削减在独特的delimination网站和众多的克隆步骤，然后进行，这往往是一个耗时和令人沮丧的process.ResultsWe已经开发出一个一步克隆方法插入两个臂到KO载体使用核酸外切酶III。我们修饰了腺相关病毒KO穿梭载体（pTK-LoxP-NEO-AAV）以产生pAAV-LIC，其在两个多克隆位点处包含两个盒。用EcoRV消化载体，得到两个片段。通过聚合酶链反应扩增与载体片段末端有16个碱基重叠的两个同源臂。纯化后，将四个片段混合，用核酸外切酶III处理，然后转化大肠杆菌，获得所需的克隆。利用该方法构建了SirT 1和HDAC 2 KO载体，并分别建立了大肠癌细胞系HCT 116的SirT 1 KO细胞和大肠癌细胞系DLD 1的HDAC 2 KO细胞。
BackgroundGene targeting is a powerful method that can be used for examining the functions of genes. Traditionally, the construction of knockout (KO) vectors requires an amplification step to obtain two homologous, large fragments of genomic DNA. Restriction enzymes that cut at unique recognitions sites and numerous cloning steps are then carried out; this is often a time-consuming and frustrating process.ResultsWe have developed a one-step cloning method for the insertion of two arms into a KO vector using exonuclease III. We modified an adeno-associated virus KO shuttle vector (pTK-LoxP-NEO-AAV) to yield pAAV-LIC, which contained two cassettes at the two multiple-cloning sites. The vector was digested withEcoRV to give two fragments. The two homologous arms, which had an overlap of 16 bases with the ends of the vector fragments, were amplified by polymerase chain reaction. After purification, the four fragments were mixed and treated with exonuclease III, then transformed intoEscherichia colito obtain the desired clones. Using this method, we constructed SirT1 and HDAC2 KO vectors, which were used to establish SirT1 KO cells from the colorectal cancer cell line (HCT116) and HDAC2 KO cells from the colorectal cancer cell line (DLD1).ConclusionsOur method is a fast, simple, and efficient technique for cloning, and has great potential for high-throughput construction of KO vectors.
登录
查看更多内容
影响因子：
14.9
作者：
Gibson DG
通讯作者：
Gibson DG
用户融合：一种快速有效的方法，用于同时融合和克隆多个PCR产品。
影响因子：
14.9
作者：
Geu-Flores, Fernando;Nour-Eldin, Hussam H.;Nielsen, Morten T.;Halkier, Barbara A.
通讯作者：
Halkier, Barbara A.
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
影响因子：
46.9
作者：
Hirata, R;Chamberlain, J;Russell, DW
通讯作者：
Russell, DW
影响因子：
48
作者：
Gibson, Daniel G.;Smith, Hamilton O.;Merryman, Chuck
通讯作者：
Merryman, Chuck