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.
Highlights
Sequencing the genome of humans and rodents has provided an immense set of uncharacterized genes, and within the past decades several genetic approaches have been taken in order to address their function
To facilitate and accelerate gene targeting to a defined locus, we modified the well-defined Rosa26 locus for fast, easy and specific integration of various transgenic constructs in mouse BALB/c Embryonic stem (ES) cells
By means of homologous recombination, we introduced a cassette that enables site-directed recombinasemediated cassette exchange (RMCE) mediated by Cre recombinase (Cre-RMCE) (Figure 1A)
Summary
Sequencing the genome of humans and rodents has provided an immense set of uncharacterized genes, and within the past decades several genetic approaches have been taken in order to address their function. In order to complement data gained from loss-of-function approaches, in vivo gain-of-function experiments have been carried out by generating mice overexpressing a gene of interest. Gain-offunction mouse models have been mainly generated by pronuclear microinjection [5] and random integration of the transgene into the genome. This quite often results in variable copy numbers, unpredictable expression profiles and sometimes gene silencing effects, requiring extensive characterization of several independent transgenic lines [6]. Targeting a single-copy transgene to a specific and well-defined locus can minimize these problems and provide a predictable and reproducible expression profile
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