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Construction of a lentiviral T/A vector for direct analysis of PCR-amplified promoters

文献类型： 外文期刊

作者： Yu, Fu-xian 1 ; Zhu, Zhi-wei 1 ; Chen, Xiao-yu 1 ; Huang, Jing 1 ; Shi, Tuan-yuan 1 ; Li, Jun-xing 1 ; Pan, Jian-zhi 1 ;

作者机构： 1.Zhejiang Acad Agr Sci, Inst Anim Husb & Vet Sci, Hangzhou, Zhejiang, Peoples R China

关键词： Lentiviral vector;T/A cloning;Promoter activity;PCR amplified product

期刊名称：MOLECULAR BIOLOGY REPORTS （ 影响因子：2.316； 五年影响因子：2.357 ）

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收录情况： SCI

摘要： The promoter plays an important role in the regulation of gene expression. To analyze a promoter's activity, we developed a novel lentiviral T/A vector that contains two reporter genes, a luciferase (Luc2) gene and a green fluorescent protein (Venus) gene, that are linked via an internal ribosome entry site (IRES2). To test the performance of this vector, phosphoglycerate kinase-1 (PGK) and elongation factor-1 alpha (EF1 alpha) promoters were amplified by PCR and inserted into this lentiviral T/A vector using T4 DNA ligase, yielding two promoter-reporter vectors: pLent-T-PGK and pLent-T-EF1 alpha. When these vectors were transfected into 293T cells, we observed a higher level of Venus expression under a fluorescence microscopy in the case of pLent-T-EF1 alpha as compared to pLent-T-PGK. The results of the luciferase reporter assay showed that the ratio of the promoter activities of EF1 alpha and PGK was approximately 9:1. The two promoter-reporter vectors were also packaged as lentiviral particles to conduct promoter activity assay in cultured cells. The ratio of the promoter activities of EF1 alpha and PGK was 4.23:1 when they were infected into 293T cells at a multiplicity of infection of 1. This value is comparable to that of a parallel experiment using the commercial luciferase reporter vector pGL4.10 with an activity ratio of 5.99:1 for EF1 alpha and PGK. These results indicate that lentiviral T/A vector will be a useful tool for analysis of promoter activity and specificity.

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