利用Red同源重组系统构建兔次黄嘌呤-鸟嘌呤磷酸核糖转移酶基因打靶载体

利用EL350基因工程菌进行同源重组，成功进行基因敲除已有报道，但利用该系统进行兔次黄嘌呤-鸟嘌呤磷酸核糖转移酶(Hypoxanthine guanine phosphoribosyl transferase，HPRT)基因突变和基因打靶方面的研究还没有报道。本实验首先在已经筛选到含有兔全长HPRT基因BAC克隆（LBNL1-304M19）的基础上，利用Red重组系统，通过Gap-Repair方式从此克隆上将一段47Kb无启动子的HPRT基因组片段（不含有第1个外显子）克隆到pBACLinkSp质粒上，产生pBACLinkSp-rHPRT质粒。然后基于pBACLinkSp-rHPRT质粒，设计不同的同源臂，从而删除了HPRT基因的不同编码区，成功构建了三个不同的HPRT基因打靶载体。同时对利用同源重组技术敲除不同大小的DNA片段的效率进行了研究。基于本实验所构建的三个不同的兔HPRT基因打靶载体，为探索兔成纤维细胞和胚胎干细胞基因打靶的适宜条件，及进一步获得兔HPRT基因敲除动物疾病模型奠定了基础。

Construction of rabbit Hypoxanthine guanine phosphoribosyl transferase gene-targeting vector by Red homologous recombination System

It has been reported that a gene can be knocked out by homologous recombination technology in EL350 genetically engineered bacteria strain. However, the study about the mutation and genetic targeting of Hypoxanthine Guanine Phosphoribosyl Transferase (HPRT) gene by this system has not been reported. In this paper, rabbit full length HPRT gene BAC clone LBNL1-304M19 is used as the template. A 47kb rabbit HPRT gene fragment, which does not have promoter and exon1, is cloned into pBACLinkSp plasmid to form pBACLinkSp-rHPRT recombinant plasmid via Gap-Repair by Red recombination system. Then, different homologous arms are designed to delete different coding region of HPRT gene on the basis of the pBACLinkSp-rHPRT plasmid. Three different HPRT gene targeting vectors have been constructed. Meanwhile, the efficiency of deleting different sizes of DNA fragment by homologous recombination technology has also been studied. These three different HPRT gene targeting vectors form the basis for exploring the gene targeting in rabbit fibroblast cells and embryonic stem cells, and making rabbit HPRT gene knockout models in the future.