利用PCR产物快速构建免HPRT基因无启动子打靶载体

目的探讨构建兔HPRT基因打靶载体的方法，为下一步获得兔HPRT基因敲除动物模型和转基因兔奠定基础。方法首先在已经筛选到含有兔全长次黄嘌呤-鸟嘌呤磷酸核糖转移酶（Hypoxanthine guanine phosphoribosyl transferase，HPRT）基因BAC克隆（LBNL1-304M19）的基础上，利用Red重组系统，从此克隆上将一段12．5kb无启动子的HPRT基因组片段克隆到pKS-质粒上，产生pKS—rHPRT质粒。然后基于pKS-rHPRT和pIGCN21质粒，通过PCR的方法获得两个不同的带有50bp HPRT基因同源序列的IRES-eGFPCre—Frt／Neo／Frt同源重组片段，最终构建HPRT基因敲除打靶载体和无启动子基因敲入型打靶载体。结果PCR、限制性内切酶及DNA序列测定，表明载体构建成功。结论成功快速地构建了HPRT基因敲除打靶载体和无启动子基因敲入型打靶载体。同时对利用同源重组技术敲除或插入DNA片段的效率进行了研究。

The Rapid Construction of Rabbit HPRT Promoterless Gene-targeting Vector with PCR Product

Objective To construct a knockout HPRT targeting vector and a promoterless knockin targeting vector,which can be used for making rabbit HPRT gene knockout models and transgenic rabbit in the future. Methods The rabbit full length HPRT gene BAC clone LBNL1-304M19 is used as the template. A 12.5kb rabbit HPRT gene fragment, which does not include promoter, is cloned into pKS- plasmid to form pKS-rHPRT recombinant plasmid via Gap-Repair by Red recombination system. Then, the PCR was used to obtain the IRES-eGFPCre-Frt/Neo/Frt fragments flanking with 50bp homologous arms for homologous recombination of rabbit HPRT gene on the basis of pKS-rHPRT and pIGCN21 plasmids. A knockout HPRT targeting vector by replacing the last three exons with a IRES-eGFPCre-Frt/ Neo/Frt cassette and a promoterless knockin targeting vector by inserting a IRES-eGFPCre-Frt/Neo/Frt cassette in the 3＇UTR of the HPRT gene were rapidly constructed. Results The identification of the vectors with PCR,enzyme restriction and sequence showed two vectors were constructed successfully. Conclusions We rapidly constructed a knockout HPRT targeting vector and a promoterless knockin targeting vector, the efficiency of deleting or inserting DNA fragment by homologous recombination technology has also been studied.