A one-step approach to obtain cell clones expressing tetracycline- responsive transactivators
Despite the wide application of the tetracycline-regulated gene expression system, several drawbacks in establishing the system in in vitro-cultured cells have been described. Most of the problems are related to the obtainment of a reliable tetracycline-regulated cell clone, which often results in arduous labor. We describe here a new approach to facilitate the screening and selection of such cell clones. We have constructed a tetracycline-responsive plasmid that harbors an antibiotic resistance gene fused to the enhanced green fluorescent protein (EGFP) gene and the luciferase gene, both under the control of a bidirectional promoter. We demonstrate that the selection of tetracycline-regulated clones is highly simplified by using this plasmid. Only clones expressing the system in a functional manner are able to survive under antibiotic selection. In addition, a quick characterization of the responsiveness of the clones is possible by monitoring GFP expression in vivo. © 2004 Elsevier Inc. All rights reserved.
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Format: | journal article biblioteca |
Language: | English |
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Elsevier
2004
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Subjects: | Tetracycline, Inducible expression, tTA transactivator, rtTA transactivator, |
Online Access: | http://hdl.handle.net/20.500.12792/3055 http://hdl.handle.net/10261/294224 |
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dig-inia-es-10261-2942242023-02-20T10:36:38Z A one-step approach to obtain cell clones expressing tetracycline- responsive transactivators Muñoz, I. Gómez, A. Zanuy, S. Carrillo, M. Tetracycline Inducible expression tTA transactivator rtTA transactivator Despite the wide application of the tetracycline-regulated gene expression system, several drawbacks in establishing the system in in vitro-cultured cells have been described. Most of the problems are related to the obtainment of a reliable tetracycline-regulated cell clone, which often results in arduous labor. We describe here a new approach to facilitate the screening and selection of such cell clones. We have constructed a tetracycline-responsive plasmid that harbors an antibiotic resistance gene fused to the enhanced green fluorescent protein (EGFP) gene and the luciferase gene, both under the control of a bidirectional promoter. We demonstrate that the selection of tetracycline-regulated clones is highly simplified by using this plasmid. Only clones expressing the system in a functional manner are able to survive under antibiotic selection. In addition, a quick characterization of the responsiveness of the clones is possible by monitoring GFP expression in vivo. © 2004 Elsevier Inc. All rights reserved. 2023-02-20T10:36:38Z 2023-02-20T10:36:38Z 2004 journal article Analytical Biochemistry 331(1): 153-160 (2004) 0003-2697 http://hdl.handle.net/20.500.12792/3055 http://hdl.handle.net/10261/294224 10.1016/j.ab.2004.05.004 en none Elsevier |
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Tetracycline Inducible expression tTA transactivator rtTA transactivator Tetracycline Inducible expression tTA transactivator rtTA transactivator |
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Tetracycline Inducible expression tTA transactivator rtTA transactivator Tetracycline Inducible expression tTA transactivator rtTA transactivator Muñoz, I. Gómez, A. Zanuy, S. Carrillo, M. A one-step approach to obtain cell clones expressing tetracycline- responsive transactivators |
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Despite the wide application of the tetracycline-regulated gene expression system, several drawbacks in establishing the system in in vitro-cultured cells have been described. Most of the problems are related to the obtainment of a reliable tetracycline-regulated cell clone, which often results in arduous labor. We describe here a new approach to facilitate the screening and selection of such cell clones. We have constructed a tetracycline-responsive plasmid that harbors an antibiotic resistance gene fused to the enhanced green fluorescent protein (EGFP) gene and the luciferase gene, both under the control of a bidirectional promoter. We demonstrate that the selection of tetracycline-regulated clones is highly simplified by using this plasmid. Only clones expressing the system in a functional manner are able to survive under antibiotic selection. In addition, a quick characterization of the responsiveness of the clones is possible by monitoring GFP expression in vivo. © 2004 Elsevier Inc. All rights reserved. |
format |
journal article |
topic_facet |
Tetracycline Inducible expression tTA transactivator rtTA transactivator |
author |
Muñoz, I. Gómez, A. Zanuy, S. Carrillo, M. |
author_facet |
Muñoz, I. Gómez, A. Zanuy, S. Carrillo, M. |
author_sort |
Muñoz, I. |
title |
A one-step approach to obtain cell clones expressing tetracycline- responsive transactivators |
title_short |
A one-step approach to obtain cell clones expressing tetracycline- responsive transactivators |
title_full |
A one-step approach to obtain cell clones expressing tetracycline- responsive transactivators |
title_fullStr |
A one-step approach to obtain cell clones expressing tetracycline- responsive transactivators |
title_full_unstemmed |
A one-step approach to obtain cell clones expressing tetracycline- responsive transactivators |
title_sort |
one-step approach to obtain cell clones expressing tetracycline- responsive transactivators |
publisher |
Elsevier |
publishDate |
2004 |
url |
http://hdl.handle.net/20.500.12792/3055 http://hdl.handle.net/10261/294224 |
work_keys_str_mv |
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