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.

Multipotency of Adult Hippocampal NSCs In Vivo Is Restricted by Drosha/NFIB

“…0.001 (**) 0.006 (**) 0.005 (**) 0.003 (**) (Chong et al, 2008;Harfe et al, 2005;Lugert et al, 2012;Tchorz et al, 2012). All mice were maintained on a C57BL6 background and were 8-10 weeks old at the onset of the experiments.…”

Visualization of cell cycle in mouse embryos with Fucci2 reporter directed by Rosa26 promoter

“…However, probe signals in R26-Fucci2 may be more ubiquitous, as Rosa26 locus is, than those in R26p-Fucci2 generated by random transgenesis. The R26-Fucci2 reporter will be revised in future studies by co-inserting a ubiquitous promoter such as CAG (Tchorz et al, 2012) and iUBC (Griswold et al, 2011) into the Rosa26 locus to enhance its transcriptional activity and by making use of 2A peptide to express the two probes in a single Rosa26 locus . The greatest advantage of the knock-in Fucci2 line is that cell cycle behavior can be examined with the parental R26R-Fucci2 reporter in a specific population of cells by Cremediated cell-type-specific loxP recombination.…”

GABA suppresses neurogenesis in the adult hippocampus through GABAB receptors

“…, GABA GFAP::CreER T2 , mR26CS-EGFP and Hes5::GFP mice have been described elsewhere (Schuler et al, 2001;Gassmann et al, 2004;Haller et al, 2004;Hirrlinger et al, 2006;Vigot et al, 2006;Casanova et al, 2009;Lugert et al, 2010;Tchorz et al, 2012). Mice were maintained on a 12-hour day/night cycle with adequate food and water under specific-pathogen-free (SPF) conditions according to institutional regulations and under license numbers Germany) and 2537 and 2538 (Kantonales Veterinäramt, Basel).…”