title: A modified RMCE-compatible Rosa26 locus for the expression of transgenes from exogenous promoters.
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, 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.
authors.affiliation: Novartis Institute for Biomedical Research, Developmental and Molecular Pathways, Novartis Pharma AG, Basel, Switzerland.
authors.forename: Jan S
authors.initials: JS
authors.lastname: Tchorz
authors.forename: Thomas
authors.initials: T
authors.lastname: Suply
authors.forename: Iwona
authors.initials: I
authors.lastname: Ksiazek
authors.forename: Claudio
authors.initials: C
authors.lastname: Giachino
authors.forename: Dimitri
authors.initials: D
authors.lastname: Cloëtta
authors.forename: Claus-Peter
authors.initials: CP
authors.lastname: Danzer
authors.forename: Thierry
authors.initials: T
authors.lastname: Doll
authors.forename: Andrea
authors.initials: A
authors.lastname: Isken
authors.forename: Marianne
authors.initials: M
authors.lastname: Lemaistre
authors.forename: Verdon
authors.initials: V
authors.lastname: Taylor
authors.forename: Bernhard
authors.initials: B
authors.lastname: Bettler
authors.forename: Bernd
authors.initials: B
authors.lastname: Kinzel
authors.forename: Matthias
authors.initials: M
authors.lastname: Mueller
mesh_terms: D000818:Animals; D001287:Attachment Sites, Microbiological / Q000235:genetics; D053595:Embryonic Stem Cells / Q000378:metabolism; D017930:Genes, Reporter / Q000235:genetics; D056426:Genetic Loci / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D019426:Integrases / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008822:Mice, Transgenic; D016254:Mutagenesis, Insertional / Q000379:methods*; D009928:Organ Specificity / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011506:Proteins / Q000235:genetics*; D022661:RNA, Untranslated; D019076:Transgenes / Q000235:genetics*
chemical_list: C110853:Gt(ROSA)26Sor non-coding RNA, mouse; D011506:Proteins; D022661:RNA, Untranslated; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008156:Luciferases; C045073:Cre recombinase; D019426:Integrases