利用同源重组构建大鼠甘油二酯激酶γ(DGKγ)慢病毒过表达载体

文题释义： 甘油二酯激酶γ(DGKγ)：属于第Ⅰ类DGK亚型，主要分布于神经系统，参与浦肯野细胞树突的发育、突触可塑性调节、神经上皮细胞的增殖和迁移、神经元分化等功能活动。 同源重组技术：一种利用同源重组的原理，进行无缝克隆的技术。该技术无需考虑插入片段的酶切位点，可将插入片段PCR产物定向克隆至任意载体的任意位点。同源重组技术还能实现多个DNA片段的一步组装，操作简单，重组效率高。 背景：慢病毒载体作为外源性转基因载体已被广泛应用，但是大鼠甘油二酯激酶γ(diacylglycerol kinase γ，DGKγ)基因慢病毒载体未见报道。 目的：用同源重组的方法构建大鼠DGKγ慢病毒过表达载体。 方法：提取成年SD大鼠脑组织总RNA，以反转录得到的cDNA作为模板，通过PCR反应分段扩增大鼠DGKγ基因CDS区5'端1 029 bp和3'端1 362 bp，用同源重组技术将这2个片段与线性化载体进行定向连接，构建CMV-rat DGKγ-GFP慢病毒载体并进行PCR扩增及测序鉴定。经293T细胞包装后产生慢病毒，收集慢病毒感染293T细胞，荧光显微镜下观察细胞中GFP的表达并应用实时荧光定量PCR和Western blotting法检测细胞中DGKγ mRNA和蛋白的表达。 结果与结论：CMV-rat DGKγ-GFP慢病毒载体经PCR扩增和测序鉴定构建成功；经CMV-rat DGKγ-GFP慢病毒感染后的293T细胞，荧光显微镜下呈GFP阳性，实时荧光定量PCR显示DGKγ mRNA的表达较空载体组显著升高(P < 0.01)，Western blotting显示DGKγ蛋白表达较空载体组极显著升高(P < 0.001)。提示：成功构建了大鼠DGKγ慢病毒过表达载体，DGKγ在293T细胞有效高表达。 ORCID: 0000-0001-9352-3043(李蕾) 中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Construction of rat diacylglycerol kinase gamma lentivirus overexpression vector by homologous recombination

BACKGROUND:Lentiviral vectors have been widely used as exogenous transgenic vectors.However,a recombinant lentiviral vector containing rat diacylglycerol kinaseγ(DGKγ)gene has not been reported.OBJECTIVE:To construct lentiviral overexpression vector of rat DGKγby homologous recombination.METHODS:Total RNA was extracted from the brain tissue of adult Sprague-Dawley rats,and the cDNA obtained by PCR was used as a template to amplify the 5'-end 1 029 bp and the 3'-end 1 362 bp of the rat DGKγgene CDS.Then,the two homologous recombination fragments were ligated into the plasmid vector.The positive clones were confirmed by PCR and DNA sequencing.The CMV-rat DGKγ-GFP lentiviral vector and the lentiviral packaging system were co-transfected into 293T cells for virus packaging and lentivirus was collected to infect 293T cells.The expression of GFP in infected 293T cells was observed under fluorescence microscope.Real-time PCR and western blot assay were used to detect the relative expression of DGKγmRNA in infected 293T cells.RESULTS AND CONCLUSION:The results of PCR simplification and sequencing indicated that the CMV-rat DGKγ-GFP lentiviral vector was successfully constructed.In 293T cells infected with CMV-rat DGKγ-GFP lentivirus,the expression of GFP was observed under fluorescence microscope and the DGKγmRNA expression was increased significantly than that of the vector group by real-time PCR(P<0.01).Western blot assay results showed that the DGKγprotein expression of the selected GFP-positive 293T cells was increased very significantly(P<0.001).To conclude,the rat DGKγlentiviral overexpression vector has been successfully constructed and maintains high expression in 293T cells.