RRC ID 35803
Author Yu FX, Zhu ZW, Chen XY, Huang J, Shi TY, Li JX, Pan JZ.
Title Construction of a lentiviral T/A vector for direct analysis of PCR-amplified promoters.
Journal Mol Biol Rep
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.
Volume 41(11)
Pages 7651-8
Published 2014-11-1
DOI 10.1007/s11033-014-3656-x
PMID 25091945
MeSH Bacterial Proteins / genetics* Cloning, Molecular DNA Primers / genetics Genetic Vectors / biosynthesis Genetic Vectors / genetics* Lentivirus / genetics* Luciferases / genetics* Luminescent Proteins / genetics* Microscopy, Fluorescence Peptide Elongation Factor 1 Phosphoglycerate Kinase / genetics Polymerase Chain Reaction Promoter Regions, Genetic / genetics* Transfection / methods
IF 1.402
Times Cited 1
DNA material CSII-EF-MCS-IRES2-Venus (RDB04384) pCMV-VSV-G-RSV-Rev (RDB04393) pCAG-HIVgp (RDB04394).