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A Modified RMCE-Compatible Rosa26 Locus for the Expression of Transgenes from Exogenous Promoters
Generation of gain-of-function transgenic mice by targeting the Rosa26 locus has been established as an alternative to classical transgenic mice produced by pronuclear microinjection. However, targeting transgenes to the endogenous Rosa26 promoter results in moderate ubiquitous expression and is not...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3258265/ https://www.ncbi.nlm.nih.gov/pubmed/22253858 http://dx.doi.org/10.1371/journal.pone.0030011 |
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author | Tchorz, Jan S. Suply, Thomas Ksiazek, Iwona Giachino, Claudio Cloëtta, Dimitri Danzer, Claus-Peter Doll, Thierry Isken, Andrea Lemaistre, Marianne Taylor, Verdon Bettler, Bernhard Kinzel, Bernd Mueller, Matthias |
author_facet | Tchorz, Jan S. Suply, Thomas Ksiazek, Iwona Giachino, Claudio Cloëtta, Dimitri Danzer, Claus-Peter Doll, Thierry Isken, Andrea Lemaistre, Marianne Taylor, Verdon Bettler, Bernhard Kinzel, Bernd Mueller, Matthias |
author_sort | Tchorz, Jan S. |
collection | PubMed |
description | Generation of gain-of-function transgenic mice by targeting the Rosa26 locus has been established as an alternative to classical transgenic mice produced by pronuclear microinjection. However, targeting transgenes to the endogenous Rosa26 promoter results in moderate ubiquitous expression and is not suitable for high expression levels. Therefore, we now generated a modified Rosa26 (modRosa26) locus that combines efficient targeted transgenesis using recombinase-mediated cassette exchange (RMCE) by Flipase (Flp-RMCE) or Cre recombinase (Cre-RMCE) with transgene expression from exogenous promoters. We silenced the endogenous Rosa26 promoter and characterized several ubiquitous (pCAG, EF1α and CMV) and tissue-specific (VeCad, αSMA) promoters in the modRosa26 locus in vivo. We demonstrate that the ubiquitous pCAG promoter in the modRosa26 locus now offers high transgene expression. While tissue-specific promoters were all active in their cognate tissues they additionally led to rare ectopic expression. To achieve high expression levels in a tissue-specific manner, we therefore combined Flp-RMCE for rapid ES cell targeting, the pCAG promoter for high transgene levels and Cre/LoxP conditional transgene activation using well-characterized Cre lines. Using this approach we generated a Cre/LoxP-inducible reporter mouse line with high EGFP expression levels that enables cell tracing in live cells. A second reporter line expressing luciferase permits efficient monitoring of Cre activity in live animals. Thus, targeting the modRosa26 locus by RMCE minimizes the effort required to target ES cells and generates a tool for the use exogenous promoters in combination with single-copy transgenes for predictable expression in mice. |
format | Online Article Text |
id | pubmed-3258265 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-32582652012-01-17 A Modified RMCE-Compatible Rosa26 Locus for the Expression of Transgenes from Exogenous Promoters Tchorz, Jan S. Suply, Thomas Ksiazek, Iwona Giachino, Claudio Cloëtta, Dimitri Danzer, Claus-Peter Doll, Thierry Isken, Andrea Lemaistre, Marianne Taylor, Verdon Bettler, Bernhard Kinzel, Bernd Mueller, Matthias PLoS One Research Article Generation of gain-of-function transgenic mice by targeting the Rosa26 locus has been established as an alternative to classical transgenic mice produced by pronuclear microinjection. However, targeting transgenes to the endogenous Rosa26 promoter results in moderate ubiquitous expression and is not suitable for high expression levels. Therefore, we now generated a modified Rosa26 (modRosa26) locus that combines efficient targeted transgenesis using recombinase-mediated cassette exchange (RMCE) by Flipase (Flp-RMCE) or Cre recombinase (Cre-RMCE) with transgene expression from exogenous promoters. We silenced the endogenous Rosa26 promoter and characterized several ubiquitous (pCAG, EF1α and CMV) and tissue-specific (VeCad, αSMA) promoters in the modRosa26 locus in vivo. We demonstrate that the ubiquitous pCAG promoter in the modRosa26 locus now offers high transgene expression. While tissue-specific promoters were all active in their cognate tissues they additionally led to rare ectopic expression. To achieve high expression levels in a tissue-specific manner, we therefore combined Flp-RMCE for rapid ES cell targeting, the pCAG promoter for high transgene levels and Cre/LoxP conditional transgene activation using well-characterized Cre lines. Using this approach we generated a Cre/LoxP-inducible reporter mouse line with high EGFP expression levels that enables cell tracing in live cells. A second reporter line expressing luciferase permits efficient monitoring of Cre activity in live animals. Thus, targeting the modRosa26 locus by RMCE minimizes the effort required to target ES cells and generates a tool for the use exogenous promoters in combination with single-copy transgenes for predictable expression in mice. Public Library of Science 2012-01-13 /pmc/articles/PMC3258265/ /pubmed/22253858 http://dx.doi.org/10.1371/journal.pone.0030011 Text en Tchorz et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Tchorz, Jan S. Suply, Thomas Ksiazek, Iwona Giachino, Claudio Cloëtta, Dimitri Danzer, Claus-Peter Doll, Thierry Isken, Andrea Lemaistre, Marianne Taylor, Verdon Bettler, Bernhard Kinzel, Bernd Mueller, Matthias A Modified RMCE-Compatible Rosa26 Locus for the Expression of Transgenes from Exogenous Promoters |
title | A Modified RMCE-Compatible Rosa26 Locus for the Expression of Transgenes from Exogenous Promoters |
title_full | A Modified RMCE-Compatible Rosa26 Locus for the Expression of Transgenes from Exogenous Promoters |
title_fullStr | A Modified RMCE-Compatible Rosa26 Locus for the Expression of Transgenes from Exogenous Promoters |
title_full_unstemmed | A Modified RMCE-Compatible Rosa26 Locus for the Expression of Transgenes from Exogenous Promoters |
title_short | A Modified RMCE-Compatible Rosa26 Locus for the Expression of Transgenes from Exogenous Promoters |
title_sort | modified rmce-compatible rosa26 locus for the expression of transgenes from exogenous promoters |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3258265/ https://www.ncbi.nlm.nih.gov/pubmed/22253858 http://dx.doi.org/10.1371/journal.pone.0030011 |
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