Wang Jun-Ping, Zhang You-Ming
State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, College of Preventive Medicine, The Third Military Medical University, Chongqing 400038, China.
Yi Chuan. 2005 Nov;27(6):953-8.
A rapid and high efficient working system for gene-targeting vector construction was developed by using Red/ET recombination. Mediated by Red/ET recombination, the objective genomic DNA was first subcloned into the targeting vector. After insertion of a PCR amplified selectable marker gene flanked with short homology arms into the targeted position, a conventional gene knock-out targeting vector was then constructed. For conditional gene knock-out targeting vector construction, with the co-operation of Cre-loxP site-specific recombination, two rounds of Red/ET recombination was just needed. Being different from PCR and endonuclease-based gene-targeting vector construction, the homologous regions used for gene targeting can be chosen as long as possible. Furthermore, no enzyme digestion, ligation and sequencing identification were involved, so that it is very efficient and labor-saving. Several different gene-targeting vectors were successfully constructed by using this system. The establishment of this working system will accelerate the gene function studies in the post-genome stage.
利用Red/ET重组技术开发了一种快速高效的基因靶向载体构建工作系统。在Red/ET重组的介导下，首先将目标基因组DNA亚克隆到靶向载体中。在将侧翼带有短同源臂的PCR扩增选择标记基因插入到目标位置后，构建传统的基因敲除靶向载体。对于条件性基因敲除靶向载体的构建，在Cre-loxP位点特异性重组的协同作用下，仅需两轮Red/ET重组。与基于PCR和核酸内切酶的基因靶向载体构建不同，用于基因靶向的同源区域可以尽可能长地选择。此外，该方法不涉及酶切、连接和测序鉴定，因此非常高效且省力。利用该系统成功构建了几种不同的基因靶向载体。该工作系统的建立将加速后基因组时代的基因功能研究。
