Noise-Reduction and Sensitivity-Enhancement of a Sleeping Beauty-Based Tet-On System
Tetracycline-inducible systems are widely used control elements for mammalian gene expression. Despite multiple iterations to improve inducibility, their use is still compromised by basal promoter activity in the absence of tetracyclines. In a mammalian system, we previously showed that the introduc...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-09-01
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| Series: | Genes |
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| Online Access: | https://www.mdpi.com/2073-4425/13/10/1679 |
| _version_ | 1797473143410917376 |
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| author | Sarah C. Saunderson SM Ali Hosseini-Rad Alexander D. McLellan |
| author_facet | Sarah C. Saunderson SM Ali Hosseini-Rad Alexander D. McLellan |
| author_sort | Sarah C. Saunderson |
| collection | DOAJ |
| description | Tetracycline-inducible systems are widely used control elements for mammalian gene expression. Despite multiple iterations to improve inducibility, their use is still compromised by basal promoter activity in the absence of tetracyclines. In a mammalian system, we previously showed that the introduction of the G72V mutation in the rtTA-M2 tetracycline activator lowers the basal level expression and increases the fold-induction of multiple genetic elements in a long chimeric antigen receptor construct. In this study, we confirmed that the G72V mutation was effective in minimising background expression in the absence of an inducer, resulting in an increase in fold-expression. Loss of responsiveness due to the G72V mutation was compensated through the incorporation of four sensitivity enhancing (SE) mutations, without compromising promoter tightness. However, SE mutations alone (without G72V) led to undesirable leakiness. Although cryptic splice site removal from rtTA did not alter the inducible control of the luciferase reporter gene in this simplified vector system, this is still recommended as a precaution in more complex multi-gene elements that contain rtTA. The optimized expression construct containing G72V and SE mutations currently provides the best improvement of fold-induction mediated by the rtTA-M2 activator in a mammalian system. |
| first_indexed | 2024-03-09T20:11:43Z |
| format | Article |
| id | doaj.art-80a115dac22c49138777f8fa623caa28 |
| institution | Directory Open Access Journal |
| issn | 2073-4425 |
| language | English |
| last_indexed | 2024-03-09T20:11:43Z |
| publishDate | 2022-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Genes |
| spelling | doaj.art-80a115dac22c49138777f8fa623caa282023-11-24T00:14:16ZengMDPI AGGenes2073-44252022-09-011310167910.3390/genes13101679Noise-Reduction and Sensitivity-Enhancement of a Sleeping Beauty-Based Tet-On SystemSarah C. Saunderson0SM Ali Hosseini-Rad1Alexander D. McLellan2Department of Microbiology and Immunology, University of Otago, Dunedin 9016, New ZealandDepartment of Microbiology and Immunology, University of Otago, Dunedin 9016, New ZealandDepartment of Microbiology and Immunology, University of Otago, Dunedin 9016, New ZealandTetracycline-inducible systems are widely used control elements for mammalian gene expression. Despite multiple iterations to improve inducibility, their use is still compromised by basal promoter activity in the absence of tetracyclines. In a mammalian system, we previously showed that the introduction of the G72V mutation in the rtTA-M2 tetracycline activator lowers the basal level expression and increases the fold-induction of multiple genetic elements in a long chimeric antigen receptor construct. In this study, we confirmed that the G72V mutation was effective in minimising background expression in the absence of an inducer, resulting in an increase in fold-expression. Loss of responsiveness due to the G72V mutation was compensated through the incorporation of four sensitivity enhancing (SE) mutations, without compromising promoter tightness. However, SE mutations alone (without G72V) led to undesirable leakiness. Although cryptic splice site removal from rtTA did not alter the inducible control of the luciferase reporter gene in this simplified vector system, this is still recommended as a precaution in more complex multi-gene elements that contain rtTA. The optimized expression construct containing G72V and SE mutations currently provides the best improvement of fold-induction mediated by the rtTA-M2 activator in a mammalian system.https://www.mdpi.com/2073-4425/13/10/1679tetracycline inducible promotersleeping beauty transposongene regulation |
| spellingShingle | Sarah C. Saunderson SM Ali Hosseini-Rad Alexander D. McLellan Noise-Reduction and Sensitivity-Enhancement of a Sleeping Beauty-Based Tet-On System Genes tetracycline inducible promoter sleeping beauty transposon gene regulation |
| title | Noise-Reduction and Sensitivity-Enhancement of a Sleeping Beauty-Based Tet-On System |
| title_full | Noise-Reduction and Sensitivity-Enhancement of a Sleeping Beauty-Based Tet-On System |
| title_fullStr | Noise-Reduction and Sensitivity-Enhancement of a Sleeping Beauty-Based Tet-On System |
| title_full_unstemmed | Noise-Reduction and Sensitivity-Enhancement of a Sleeping Beauty-Based Tet-On System |
| title_short | Noise-Reduction and Sensitivity-Enhancement of a Sleeping Beauty-Based Tet-On System |
| title_sort | noise reduction and sensitivity enhancement of a sleeping beauty based tet on system |
| topic | tetracycline inducible promoter sleeping beauty transposon gene regulation |
| url | https://www.mdpi.com/2073-4425/13/10/1679 |
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