诱导性表达Cre重组酶转基因载体的构建及鉴定

Construction and identification of inducible Cre recombinase expression transgenic vector

背景:猪在遗传上与人类相似,因此猪是研究人类疾病最好的动物模型,而对于这方面的研究在国内外报道较少。目的:建立诱导性表达Cre重组酶的转基因载体。设计、时间及地点:单一样本观察,于2007-10/2008-05在吉林大学农学部畜牧兽医学院完成。材料:DH5a菌株E.coliDH5α,质粒pcDNA3.1(+)、PET28a(+)、pCIneo,重组质粒PMD-Cre和猪成纤维细胞由北华大学医学部基础医学院细胞生物教研室保存;重组质粒pGC-FRT2NeoRRRR和Mx1克隆载体pGL3-Mx1由意大利Stefano教授馈赠。方法:构建的载体包括以下元件:Mx1启动子用来启动Cre的表达,Cre的作用是工具便于以后做基因敲除,BGHpolyA是一个终止信号,FRT2NeoR中的FRT是FLP酶的识别位点便于切除筛选标记,NeoR是筛选标记。通过聚合酶链反应方法分别从pGL3Mx1载体上和pcDNA3.1(+)载体上扩增猪Mx1启动子和BGHpolyA。利用重叠聚合酶链反应方法获得猪源的Cre重组酶基因,并从pGCFRT2NeoR上用XholⅠandSalⅠ酶切得到FRT2NeoR盒子。将上述4个片段利用SOE-PCR及酶连接的方法用T4DNA连接酶连接,然后利用原核表达载体PET28a(+)构建出Cre表达载体Mx1-Cre-BGHpolyA-FRT2neoR。主要观察指标:Cre重组酶表载体PET28a(+)-Mx1-Cre-BGHpolyA-FRT2neoR的鉴定。结果:PET28a(+)-Mx1-Cre-BGHpolyA-FRT2neoR重组质粒经NheⅠ和NotⅠ双酶切后完全符合理论上可切出的大小片段,载体构建成功。结论:成功的构建了诱导性表达Cre重组酶表达载体Mx1-Cre-BGHpolyA-FRT2neoR。

BACKGROUND： Pigs are similar with humans in genetics, so pigs are the best model animal of human disease. This kind of research reports at home and abroad are few. OBJECTIVE： To establish induced expression of Cre transgenic vector. DESIGN, TIME AND SETTING： A single sample observation was completed in Animal Husbandry and Veterinary Institute of Agricultural School of Jilin University from October 2007 to May 2008. MATERIALS： DH5 a strain E.coliDH5 a, plasmid pcDNA3.1 （＋）, PET28a （＋）, pClneo, recombinant plasmid PMD-Cre and pig fibroblasts were stored by Department of Cellular Biology, Basic Medical College of Beihua University; recombinant plasmid pGC-FRT2NeoRRRR and Mx 1 cloning vector pGL3-Mx1 were gifts from Italy, Professor Stefano. METHODS： Construction of the carrier included the following components： Mx I promoter was used to activate the expression of Cre, the role of Cre was the tools to facilitate to knockout, BGHpolyA was a termination signal, FRT of FRT2NeoR was the identify sites of enzyme FLP for removal screening tag, NeoR was the selection marker. Through polymerase chain reaction, pig Mx 1 promoter and BGHpolyA were amplified from pGL3Mxl vector and peDNA3.1 （＋） vector. Using overlapping polymerase chain reaction method we obtained swine Cre gene, and from pGCFRT2NeoR with Xhol Ⅰ and Sal Ⅰ digested we obtained FRT2NeoR box. The above four fragments were connected together by SOE-PCR method and T4 DNA ligase connection. Then the expression vector PET28a （＋） was used to build Cre expression vector Mx 1-Cre-BGHpolyA-FRT2neoR. MAIN OUTCOME MEASURES： The identification of Cre recombinase expression vector PET28a （＋）-Mx 1 -Cre-BGHpolyA-FRT2neoR. RESULTS： PET28a （＋）-Mx1-Cre-BGHpolyA-FRT2neoR recombinant plasmid was double digested by Nhe Ⅰ and Not Ⅰ, and could be cut to the size of fragments, which was fully in line with theory. The vector construction was a success. CONCLUSION： The inducible Cre recombinase expression vector Mx1-Cre-BGHpolyA-FRT2neoR can be successfully constructed.