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

Generation of Chimaeric mice with mutations in the signaling domain of CD22 and analysis of the function of CD22 in knockout mice

Gegenstand dieser Arbeit ist die Untersuchung von Aspekten der Funktion von CD22, einem B-Zell spezifischen Transmembran-Rezeptor der Siglec-Familie (Sialinsäure-bindende Immunglobulin-ähnliche Lectine). Mit der äußersten der 7 extrazellulären Ig-ähnlichen Domänen kann CD22 spezifisch mit a2,6-Sialinsäure interagieren. In der cytoplasmatischen Domäne von CD22 befinden sich 6 konservierte Tyrosine, 3 davon in ITIMs (Immunrezeptor tyrosinhaltige inhibitorischen Motiven). Nach Kreuzvernetzung des B-Zell Rezeptors wird CD22 tyrosinphosphoryliert. Die cytosolische Tyrosin-Phosphatase SHP-1 bindet in der Folge an die phosphorylierten ITIMs, wird aktiviert, und inhibiert das BCR Ca2+-Signal. Gleichzeitig binden jedoch auch positive Modulatoren des BCR-Signals (Lyn, Syk, PLCg PI3K, und Grb-2) an CD22, deren Rolle im Zusammenhang mit CD22 bislang ungeklärt ist. 1. In einem Hauptteil der Arbeit sollten zwei Knockin Mausmodelle generiert werden. Das eine Mausmodell (CD22-ITIM-KO) sollte zerstörte ITIM-Motive enthalten. Bei dem anderen (CD22-Tailless) sollte die gesamte cytoplasmatische Domäne von CD22 fehlen. Beide Modelle sollten der Untersuchung der Rolle der an CD22 bindenden positiven Modulatoren des BCR-Signals, und des Zusammenhangs zwischen Signaltransduktion und Ligandenbindung in vivo dienen. Die Klonierung der Targeting-Vektoren für CD22-ITIM-KO (pCD22-ITIM-KO) und CD22-Tailless (pCD22-Tailless) wurde abgeschlossen. Mit Hilfe ebenfalls klonierter Kontrollvektoren wurden PCRs zur Identifizierung homolog rekombinanter ES-Zell Klone etabliert. Für beide Targeting-Konstrukte wurden nach Transfektion von C57BL/6 ES-Zellen homolog rekombinante Klone erhalten, und mittels Southern Blot und Sequenzierung der eingeführten Mutationen vollständig charakterisiert. Nach Cre/lox-vermittelter Deletion der Selektionskassette des Targeting-Konstrukts folgte Injektion voll charakterisierter CD22-ITIM-KO Klone in BALB/c-Blastozysten. Es wurden 5 chimäre Tiere erhalten, von denen keines die Mutationen durch die Keimbahn weitergab. Transfektionen der C57BL/6 Targeting-Konstrukte in anderen, nicht-isogenen ES-Zell Linien ergaben keine homologen Rekombinanten. Das Auffinden der genomischen Sequenz von CD22 in einer Internet-Datenbank ermöglichte die Verlängerung von pCD22-ITIM-KO um ca. 4 kb mit 129/ola-DNA. Eine Transfektion dieses neuen Konstruktes in eine 129/ola ES-Zelllinie ergab keine homologen Rekombinanten. Jedoch öffnet die nun bekannte genomische CD22-Sequenz den Weg zu einfacher Neukonstruktion von pCD22-ITIM-KO mit 129/ola-DNA, oder zu einer Veränderung und Verbesserung der vorhandenen C57BL/6-Vektoren. 2. Zur Untersuchung der Auswirkung der zerstörten ITIMs auf Tyrosinphosphorylierung und SHP-1 Assoziation von CD22 in vitro in einer Zelllinie wurde ein CD22-ITIM-KO-Expressionsvektor konstruiert, und Sialyltransferase/CD22-ITIM-KO Doppeltransfektanten der Plasmozytom-Zelllinie J558L gewonnen. CD22-ITIM-KO wurde nach BCR-Stimulation nicht tyrosinphosphoryliert, SHP-1 konnte entsprechend nicht mit CD22-ITIM-KO assoziieren. Die Ergebnisse zeigen die Funktionalität des CD22-ITIM-KO Konstrukts hinsichtlich ITIM-Phosphorylierung und SHP-1 Bindung. Weiterhin zeigten die Ergebnisse, daß die ITIM-Tyrosine wichtig für die Phosphorylierung der nicht-ITIM-Tyrosine sind. 3. Interaktion von CD22 mit a2,6-Sialinsäure auf der selben Zelloberfläche (in Cis) spielt eine wichtige Rolle bei der Zell-Zell-Interaktion und bei der intrazellulären Signaltransduktion. In dieser Arbeit wurden erstmals mittels Durchflußcytometrie B-Zellen mit CD22, dessen Liganden-Bindungsstelle nicht durch endogene a2,6-Sialinsäure besetzt ist (demaskiertes CD22), identifiziert. Ca. 10,5% aller B220+ Milzzellen von Wildtyp-Mäusen, aber nur ca. 4,5% der B220+ Milzzellen aus CD22-/- Mäusen waren in der Lage, exogene a2,6-Sialinsäure zu binden. Dieser Effekt ist zum Großteil auf CD22 zurückzuführen. Genauere FACS-Analyse zeigte, daß Zellen mit demaskiertem CD22 in der Fraktion der Transitionalen B-Zellen Typ 2 (T2-Zellen) angereichert sind, und Zeichen von Aktivierung (B7.2, CD25, CD69) zeigen. In Übereinstimmung damit führte in vitro Aktivierung von B-Zellen mit LPS oder IL4 zu CD22-abhängiger Demaskierung. 4. FACS-Färbungen zeigten, daß das Marginalzonen (MZ) B-Zell Kompartiment in CD22-/- Mäusen um ca. 70-80% gegenüber wt-Mäusen verkleinert ist. In Bestätigung früherer Arbeiten war die Immunantwort gegen i.p.-injizierte Thymusunabhängige Antigene Typ 2 (TI2-Antigene) in CD22-/- Mäusen 2-fach reduziert. Die Antwort war jedoch signifikant stärker reduziert (3-4-fach), wenn die gleiche Antigen-Menge i.v.-injiziert wurde, eine Situation, in der bevorzugt die MZ B-Zellen der Milz in Kontakt mit im Blutstrom transportierten Antigenen kommen. Es ist wahrscheinlich, daß die bekannte Defizienz in TI2-Immunantworten in CD22-/- Mäusen auf die verringerte MZ B-Zell Anzahl zurückzuführen ist.Aim of this thesis was to investigate aspects of the function of CD22, a B-cell specific member of the Siglec-family (sialic acid binding immunoglobulin–like lectins). CD22 can specifically bind a2,6-sialic acid with the outermost of its 7 extracellular Ig-domains. There are 6 conserved tyrosine residues in the cytoplasmic domain, 3 of which lie within ITIMs (immunoreceptor tyrosine-based inhibitory motifs). Following BCR engagement, CD22 becomes tyrosine-phosphorylated. Consecutively, the cytosolic tyrosin-phosphatase SHP-1 binds to the phosphorylated ITIMs, becomes activated and inhibits the BCR-signal. There are also some positive modulators of the BCR-signal which bind to CD22 (Lyn, Syk, PLCg PI3K, und Grb-2). The role of these molecules in the context of CD22 remains to be elucidated. 1. One main goal of this work was the generation of two new knockin mouse lines. In the first line (CD22-ITIM-KO), the ITIMs of CD22 were to be destroyed. A second line in which CD22 lacks its cytoplasmic tail was to be generated (CD22-Tailless). Both models were supposed to serve the in vivo investigation of the role of the positive modulators of the BCR-signal and the interrelation of CD22 signaling and ligand binding. Cloning of the targeting vectors pCD22-ITIM-KO and pCD22-tailless was completed. Using control vectors, which were also cloned, screening-PCRs for the identification of homologous recombined ES-cell clones were established. C57BL/6 ES-cells were transfected with the targeting constructs, and homologous recombinants were identified with PCR and Southern blot. The introduced mutations were confirmed by sequencing. Following cre/lox-mediated deletion of the selection cassette, fully characterised CD22-ITIM-KO clones were injected into BALB/c-blastocysts. Five chimaeras were obtained, none of which transmitted the mutations through the germline. No homologous recombinants were obtained upon injection of the C57BL/6 targeting constructs into other, non-isogenic ES-cell lines. Finding of the genomic sequence of murine CD22 in a database made it possible to extend pCD22-ITIM-KO by 4 kb with DNA from a 129/ola mouse strain. The new construct was used to transfect 129/ola ES-cells, which are generally known to be kariotypically more stable than C57BL/6 ES-cells. No homologous recombinants were obtained. The now known sequence of the CD22 gene will make it possible to reconstruct pCD22-ITIM-KO based on 129-DNA or to modify and improve the already existing C57BL/6-constructs. 2. To investigate the consequences of the destroyed ITIMs on tyrosine-phosphorylation and SHP-1 association of CD22 in vitro, a CD22-ITIM-KO expression-vector was constructed and sialyl-transferase/CD22-ITIM-KO double-transfectants of the murine plasmocytoma-line J558L were generated. CD22 tyrosine-phosphorylation after BCR-stimulation was completely abolished in CD22-ITIM-KO transfectants. Accordingly, SHP-1 couldnt associate with CD22-ITIM-KO. The results prove the funcionality of the CD22-ITIM-KO construct with respect to ITIM-phosphorylation and binding of SHP-1. Furthermore the results show that the ITIM-tyrosines are also important for the phosphorylation of the non-ITIM-tyrosines. 3. Interaction of CD22 with its ligand a2,6-sialic acid on the surface of the same cell (in cis) plays an important role in cell-cell-interaction and intracellular signal transduction. In this work B-cells on which the ligand binding site of CD22 is not occupied by endogenous a2,6-sialic acid (demasked CD22) were identified by FACS for the first time. 10,5% of all B220+ spleen cells from wild type mice, in contrast to only 4,5% B220+ spleen cells from CD22-/- mice were able to bind exogenous a2,6-sialic acid. This effect is mainly due to the presence or absence of CD22, respectively. Detailed FACS-analyses revealed that cells with demasked CD22 are enriched in the fraction of the transitional B-cells type 2 (T2 cells) and show signs of activation. Accordingly, in vitro activation of B-cells with LPS or IL4 resulted in CD22-dependent demasking. 4. FACS-analyses showed that the marginal zone (MZ) B-cell compartment in CD22-/- mice is strongly reduced (by about 70-80%). Earlier results showing that the immune response against i.p.-injected TI2-antigens is about 2-fold reduced in CD22-/- mice could be confirmed. However, the response was significantly more impaired (3-4-fold) when the same dose of antigen was applied i.v., a situation where preferentially the MZ B-cells of the spleen come in contact with antigen carried along with the blood flow. The known deficiency in TI2-responses in CD22-/- mice is likely due to non-sufficient MZ B-cell numbers in these animals

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

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

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 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