Abstract
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-1α (EF1α) 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-EF1α. 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-EF1α as compared to pLent-T-PGK. The results of the luciferase reporter assay showed that the ratio of the promoter activities of EF1α 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 EF1α 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 EF1α 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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Acknowledgments
This work was supported by the Major Science and Technology Project of Zhejiang Province (Grant 2010C12006, 2012C12906-8-1), the Natural Science Foundation of Zhejiang Province (Grant LQ12C17003).
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Yu, Fx., Zhu, Zw., Chen, Xy. et al. Construction of a lentiviral T/A vector for direct analysis of PCR-amplified promoters. Mol Biol Rep 41, 7651–7658 (2014). https://doi.org/10.1007/s11033-014-3656-x
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DOI: https://doi.org/10.1007/s11033-014-3656-x