論文 - 詳細
| RRC ID | 35803 |
|---|---|
| 著者 | Yu FX, Zhu ZW, Chen XY, Huang J, Shi TY, Li JX, Pan JZ. |
| タイトル | Construction of a lentiviral T/A vector for direct analysis of PCR-amplified promoters. |
| ジャーナル | 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. |
| 巻・号 | 41(11) |
| ページ | 7651-8 |
| 公開日 | 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 |
| 引用数 | 1 |
| WOS 分野 | BIOCHEMISTRY & MOLECULAR BIOLOGY |
| オルトメトリクス指標 |
オルトメトリクス指標項目
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| 最多言及媒体 | X(Twitter) |
| 各媒体での言及数の合計 | 1 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 0.0 |
| リソース情報 | |
| 遺伝子材料 | CSII-EF-MCS-IRES2-Venus (RDB04384) pCMV-VSV-G-RSV-Rev (RDB04393) pCAG-HIVgp (RDB04394). |