| 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
|
|
WOS Category
|
BIOCHEMISTRY & MOLECULAR BIOLOGY
|
| Resource |
| DNA material |
CSII-EF-MCS-IRES2-Venus (RDB04384)
pCMV-VSV-G-RSV-Rev (RDB04393)
pCAG-HIVgp (RDB04394). |
