A modified RMCE-compatible Rosa26 locus for the expression of transgenes from exogenous promoters
JournalArticle (Originalarbeit in einer wissenschaftlichen Zeitschrift)
 
ID 1195136
Author(s) Tchorz, J. S.; Suply, T.; Ksiazek, I.; Giachino, C.; Cloetta, D.; Danzer, C. P.; Doll, T.; Isken, A.; Lemaistre, M.; Taylor, V.; Bettler, B.; Kinzel, B.; Mueller, M.
Author(s) at UniBasel Bettler, Bernhard
Taylor, Verdon
Year 2012
Title A modified RMCE-compatible Rosa26 locus for the expression of transgenes from exogenous promoters
Journal PLoS ONE
Volume 7
Number 1
Keywords Animals; Attachment Sites, Microbiological/genetics; Embryonic Stem Cells/metabolism; Genes, Reporter/genetics; Genetic Loci/*genetics; Green Fluorescent Proteins/metabolism; Integrases/*metabolism; Luciferases/metabolism; Mice; Mice, Inbred BALB C; Mice, Transgenic; Mutagenesis, Insertional/*methods; Organ Specificity/genetics; Promoter Regions, Genetic/*genetics; Proteins/*genetics; Transgenes/*genetics
Abstract 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, EF1? and CMV) and tissue-specific (VeCad, ?SMA) 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.
Publisher Public Library of Science
ISSN/ISBN 1932-6203
edoc-URL http://edoc.unibas.ch/dok/A6005322
Full Text on edoc No
Digital Object Identifier DOI 10.1371/journal.pone.0030011
PubMed ID http://www.ncbi.nlm.nih.gov/pubmed/22253858
ISI-Number WOS:000301361500030
Document type (ISI) Article
 
   

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