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 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

Tissue-restricted gene expression from the modRosa26^FRT locus.

<p>A) Scheme depicting the generation of the modRosa26^FRT locus in ES cells. A Stop sequence and a HygR selection cassette flanked by heterospecific FRT sites (FRT3 and FRTwt) were targeted to the Rosa26 locus between exons 1 and 2 by homologous recombination. After successful recombination, the Stop cassette is located downstream of the endogenous Rosa26 promoter (B) Flp-RMCE was performed by introducing the VeCad or αSMA promoter driving Cre recombinase into modRosa26^FRT ES cells, which were then used to generate transgenic mice (mR26-VeCad-Cre or mR26-αSMA-Cre mice). The primers and probe for genotyping are indicated (1–3). Both lines were crossed with ActB-EGFP reporter mice to monitor Cre expression, yielding mR26-VeCad-Cre/ActB-EGFP and mR26-αSMA-Cre/ActB-EGFP mice. (C) Immunostaining for EGFP in mR26-αSMA-Cre/ActB-EGFP mice shows αSMA promoter activity almost exclusively in smooth muscle cells, as seen in lung, stomach and intestine. Few EGFP stained intestinal epithelial cells indicate ectopic SMA promoter activity (asterisks) (D) Immunostaining for EGFP in mR26-VeCad-Cre/ActB-EGFP mice reveals VeCad promoter activity mostly restricted to endothelial cells, as seen in lung, brain and kidney. Some EGFP stained epithelial cells in kidney also indicate ectopic VeCad-Cre expression (asterisks).</p

Comparison of different ubiquitous promoters in the modRosa26^LoxP locus.

<p>(A) Organs from transgenic mice showing different intensity of overall EGFP fluorescence under low-magnification microscopy, using the same conditions. ActB mice were used as controls for high EGFP expression levels. The pCAG promoter shows the highest overall fluorescence. The CMV promoter showed strong activity only in testis. (B) EGFP fluorescence was quantified in organ homogenates from mR26-pCAG-EGFP, mR26-EF1α-EGFP, mR26-CMV-EGFP and ActB mice. In most organs, EGFP fluorescence was highest in mR26-pCAG-EGFP mice, except fat tissue where ActB mice showed higher levels. Heart, pancreas and muscle showed extremely high levels of EGFP fluorescence. Background fluorescence determined in organ homogenates from wt mice was subtracted (n≥3 mice per genotype). Values are shown as mean ± SEM. (C) Exemplary pictures from cryosections showing strong and ubiquitous EGFP expression in muscle (costained with wheat germ agglutinin (WGA) for muscle fiber wall and DAPI) and brain (hippocampus, costained with NeuN for neurons and GFAP for astrocytes). Mosaic EGFP fluorescence was detected in livers of mR26-pCAG-EGFP mice (costained with HNF4α for hepatocytes and DBA for bile ducts). Magnified insets show both EGFP+ and EGFP- hepatocytes.</p

Generation of luciferase reporter mice (mR26CS-Luc).

<p>(A) Flp-RMCE into the modRosa26^FRT locus was performed, replacing the HygR in the modRosa26^FRT ES cells with the FRT/FRTwt-flanked sequence from the Flp-RMCE targeting plasmid. The Flp-RMCE targeting plasmid contains the pCAG promoter followed by a LoxP-flanked (floxed) STOP cassette (STOP2), the luciferase cDNA and a NeoR cassette, flanked as a group by FRT3/FRTwt sites. Successfully targeted ES cells were used to generate mR26CS-Luc mice. After crossing mR26CS-Luc mice with Cre mice, ubiquitous or tissue-restricted luciferase reporter expression can be obtained to monitor reporter expression in living mice upon Luciferin injection. Using Xenogen imaging, adult mR26CS-Luc/CMV-Cre mice show ubiquitous luciferase activity throughout the body (B), while mR26CS-Luc/AlbCre mice show luciferase activity restricted to the liver upon Luciferin injection (C). Luciferin-injected mR26SC-Luc control mice never showed luciferase activity.</p

Generation of mice with a modified Rosa26 locus (modRosa26^LoxP) and testing of different promoters.

<p>(A) Scheme depicting the generation of the modRosa26^LoxP in ES cells. A Stop sequence and a HygR selection cassette flanked by heterospecific LoxP sites (Lox511 and LoxP) were targeted to the Rosa26 locus between exons 1 and 2 by homologous recombination. After successful recombination, the Stop cassette is located downstream of the endogenous Rosa26 promoter. (B) Cre-RMCE into the modRosa26^LoxP locus. In the Cre-RMCE targeting plasmid, a promoter, EGFP and an FRT-flanked neomycin selection cassette (NeoR) were flanked by heterospecific LoxP sites (Lox511 and LoxP) as a group. Cre-RMCE was used to replace the HygR in the modRosa26^LoxP ES cells with the Lox511/LoxP-flanked sequence in the RMCE targeting plasmid. A pCAG, CMV or EF1α promoter driving EGFP was introduced. Insertion of EGFP without any promoter (NoP) controls for functional shielding of the integration site from the endogenous Rosa26 promoter. Binding regions for TaqMan genotyping primers (1, 3) and probe (2), primers for checking integration into the modRosa26^LoxP locus (4–7) and the Southern hybridization probe, as well as the BamHI sites used for Southern blot analysis are indicated. (C) Southern blot analysis on genomic DNA-derived ES cell lines shows specific integration into the modRosa26^LoxP locus. A 2.4-kb BamHI fragment was detected using a Neo probe. mR26-pCAG-EGFP, mR26-CMV-EGFP, mR26-EF1α-EGFP and mR26-NoP-EGFP ES cells show successful targeting of the modRosa26^LoxP locus, without additional integrations at random sites. Wt ES cells show no signal. (D) Modified ES cells showing strong EGFP fluorescence. The pCAG, EF1α and CMV promoters drive strong EGFP expression in the modRosa26^LoxP in vitro. ES cells without promoter but with EGFP inserted into the modRosa26^LoxP locus do not show EGFP fluorescence, indicating functional shielding of the integration site from the endogenous Rosa26 promoter. (E) FACS analysis in mR26-NoP-EGFP ES cells showed no EGFP fluorescence when compared to wt ES cells. mR26-EF1α-EGFP ES cells showed the highest EGFP fluorescence, followed by mR26-pCAG-EGFP, ActB and mR26-CMV-EGFP ES cells.</p

Generation of the modRosa26^FRT locus and a reporter strain for strong EGFP expression.

<p>(A) Flp-RMCE into the modRosa26^FRT locus, replacing the HygR selection cassette in the modRosa26^FRT ES cells with an FRT/FRTwt-flanked sequence in the Flp-RMCE targeting plasmid. The Flp-RMCE targeting plasmid contains the pCAG promoter followed by a LoxP-flanked (floxed) STOP cassette, the EGFP cDNA and a NeoR cassette, flanked as a group by FRT/FRTwt sites. Successfully targeted ES cells were used to generate mR26-CS-EGFP mice. After crossing mR26-CS-EGFP mice with several Cre mice, ubiquitous or tissue-restricted EGFP reporter expression could be obtained. (B) mR26CS-EGFP/CMV-Cre E12.5 embryos show ubiquitous EGFP expression, while mR26CS-EGFP/Nestin-Cre mice show EGFP expression restricted to the brain and neural tube. EGFP expression in E10.5 mR26CS-EGFP/Myf5-Cre embryos was restricted to the somites (magnified inset), limbs and parts of the brain. (C) Neural stem cells isolated from E14.5 mR26CS-EGFP/Nestin-Cre mice form neurospheres with ubiquitous and strong EGFP fluorescence, while mR26CS-EGFP mice show no fluorescence. These cells were subsequently differentiated (lower panels), showing strong EGFP fluorescence in mR26CS-EGFP/Nestin-Cre-derived cells (counterstained with DAPI, the neuronal marker Tuj1 and the glial marker GFAP). (D) Adult mR26CS-EGFP/Glast-CreERT2 mice show strong and specific EGFP fluorescence in astrocytes and in the adult neural stem cell niche (asterisks) upon tamoxifen administration. EGFP+ adult neural stem cells are present in the subgranular zone (SGZ) of the dentate gyrus and the subventricular zone (SVZ). Recombined EGFP+ cells from the SVZ can be traced through the rostral migratory stream into the olfactory bulb. NeuN stains for mature neurons (blue) and BLBP for adult neural stem cells and astrocytes (red). Note: no EGFP-antibody staining was used in C–E, since the mR26CS-EGFP reporter mouse offers very high EGFP expression levels which can be easily detected without any staining.</p