Endogenous ROSA26 Promoter Research Articles

IL-22 fate reporter reveals origin and control of IL-22 production in homeostasis and infection.

IL-22 is a cytokine that regulates tissue homeostasis at barrier surfaces. A variety of IL-22-producing cell types is known, but identification on the single-cell level remains difficult. Therefore, we generated a fate reporter mouse that would allow the identification of IL-22-producing cells and their fate mapping in vivo. To trace IL-22-expressing cells, a sequence encoding Cre recombinase was cloned into the Il22 locus, and IL22(Cre) mice were crossed with reporter mice expressing enhanced yellow fluorescence protein (eYFP) under control of the endogenous Rosa26 promoter. In IL22(Cre)R26R(eYFP) mice, the fluorescent reporter permanently labels cells that have switched on Il22 expression, irrespective of cytokine production. Despite a degree of underreporting, eYFP expression was detectable in nonimmune mice and restricted to group 3 innate lymphoid cells (ILC3) in the gut and γδ T cells in skin or lung. Upon skin challenge with imiquimod, eYFP(+) γδ and CD4 T cells expanded in the skin. Infection with Citrobacter rodentium initially was controlled by ILC3, followed by expansion of eYFP(+) CD4 T cells, which were induced in innate lymphoid follicles in the colon. No eYFP expression was detected in small intestinal Th17 cells, and they did not expand in the immune response. Colonic eYFP(+) CD4 T cells exhibited plasticity during infection with expression of additional cytokines, in contrast to ILC3, which remained largely stable. Single-cell quantitative PCR analysis of eYFP(+) CD4 T cells confirmed their heterogeneity, suggesting that IL-22 expression is not confined to particular subsets or a dedicated Th22 subset.

Conditional and constitutive expression of a Tbx1-GFP fusion protein in mice

BackgroundVelo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS) is caused by a 1.5-3 Mb microdeletion of chromosome 22q11.2, frequently referred to as 22q11.2 deletion syndrome (22q11DS). This region includes TBX1, a T-box transcription factor gene that contributes to the etiology of 22q11DS. The requirement for TBX1 in mammalian development is dosage-sensitive, such that loss-of-function (LOF) and gain-of-function (GOF) of TBX1 in both mice and humans results in disease relevant congenital malformations.ResultsTo further gain insight into the role of Tbx1 in development, we have targeted the Rosa26 locus to generate a new GOF mouse model in which a Tbx1-GFP fusion protein is expressed conditionally using the Cre/LoxP system. Tbx1-GFP expression is driven by the endogenous Rosa26 promoter resulting in ectopic and persistent expression. Tbx1 is pivotal for proper ear and heart development; ectopic activation of Tbx1-GFP in the otic vesicle by Pax2-Cre and Foxg1-Cre represses neurogenesis and produces morphological defects of the inner ear. Overexpression of a single copy of Tbx1-GFP using Tbx1Cre/+ was viable, while overexpression of both copies resulted in neonatal lethality with cardiac outflow tract defects. We have partially rescued inner ear and heart anomalies in Tbx1Cre/- null embryos by expression of Tbx1-GFP.ConclusionsWe have generated a new mouse model to conditionally overexpress a GFP-tagged Tbx1 protein in vivo. This provides a useful tool to investigate in vivo direct downstream targets and protein binding partners of Tbx1.

Dissecting IL-22 biology using a fate reporter mouse (P6263)

Abstract Interleukin-22 is a cytokine that regulates tissue homeostasis at barrier surfaces. A number of IL-22 producing cell types has been identified, but questions remain regarding their stability and relationship to other subsets. We therefore generated a fate reporter mouse that would allow not only the identification of IL-22 producing cells but also their fate mapping in vivo. To trace cells expressing IL-22, a sequence encoding Cre recombinase was cloned into the Il22 locus (Il22Cre). To visualize Cre activity, the IL22Cre mice were then crossed with reporter mice expressing enhanced yellow fluorescence protein (eYFP) under the control of the endogenous Rosa26 promoter (R26ReYFP). In IL22CreR26ReYFP mice, the fluorescent reporter permanently labels cells that have switched on Il22 expression regardless of the present production status of this cytokine. Using this mouse strain we show that IL-22 expression is restricted to immune cells, the main producers being innate lymphoid cells in gut and γδT cells in skin or lung in an unchallenged mouse. In a model of imiquimod induced psoriasiform skin inflammation we observed a substantial accumulation of IL22-eYFP+ cells, demonstrating the functionality of our reporter in vivo. Further studies are aimed at investigating the role of IL-22-producing cells in infectious, inflammatory and autoimmune diseases and to delineate their relationship with other cytokine producing cell subsets, notably innate lymphoid cells.

The ROSA26-iPSC Mouse: A Conditional, Inducible, and Exchangeable Resource for Studying Cellular (De)Differentiation

Control of cellular (de)differentiation in a temporal, cell-specific, and exchangeable manner is of paramount importance in the field of reprogramming. Here, we have generated and characterized a mouse strain that allows iPSC generation through the Cre/loxP conditional and doxycycline/rtTA-controlled inducible expression of the OSKM reprogramming factors entirely from within the ROSA26 locus. After reprogramming, these factors can be replaced by genes of interest-for example, to enhance lineage-directed differentiation-with the use of a trap-coupled RMCE reaction. We show that, similar toESCs, Dox-controlled expression of the cardiac transcriptional regulator Mesp1 together with Wnt inhibition enhances the generation of functional cardiomyocytes upon invitro differentiation of such RMCE-retargeted iPSCs. This ROSA26-iPSC mouse model is therefore an excellent tool for studying both cellular reprogramming and lineage-directed differentiation factors from the same locus and will greatly facilitate the identification and ease of functional characterization of the genetic/epigenetic determinants involved in these complex processes.

Bmi1 Confers Resistance to Oxidative Stress on Hematopoietic Stem Cells

BackgroundThe polycomb-group (PcG) proteins function as general regulators of stem cells. We previously reported that retrovirus-mediated overexpression of Bmi1, a gene encoding a core component of polycomb repressive complex (PRC) 1, maintained self-renewing hematopoietic stem cells (HSCs) during long-term culture. However, the effects of overexpression of Bmi1 on HSCs in vivo remained to be precisely addressed.Methodology/Principal findingsIn this study, we generated a mouse line where Bmi1 can be conditionally overexpressed under the control of the endogenous Rosa26 promoter in a hematopoietic cell-specific fashion (Tie2-Cre;R26StopFLBmi1). Although overexpression of Bmi1 did not significantly affect steady state hematopoiesis, it promoted expansion of functional HSCs during ex vivo culture and efficiently protected HSCs against loss of self-renewal capacity during serial transplantation. Overexpression of Bmi1 had no effect on DNA damage response triggered by ionizing radiation. In contrast, Tie2-Cre;R26StopFLBmi1 HSCs under oxidative stress maintained a multipotent state and generally tolerated oxidative stress better than the control. Unexpectedly, overexpression of Bmi1 had no impact on the level of intracellular reactive oxygen species (ROS).Conclusions/SignificanceOur findings demonstrate that overexpression of Bmi1 confers resistance to stresses, particularly oxidative stress, onto HSCs. This thereby enhances their regenerative capacity and suggests that Bmi1 is located downstream of ROS signaling and negatively regulated by it.

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.

A Comparison of Exogenous Promoter Activity at the ROSA26 Locus Using a PhiC31 Integrase Mediated Cassette Exchange Approach in Mouse ES Cells

The activities of nine ubiquitous promoters (ROSA26, CAG, CMV, CMVd1, UbC, EF1α, PGK, chicken β-actin and MC1) have been quantified and compared in mouse embryonic stem cells. To avoid the high variation in transgene expression which results from uncontrolled copy number and chromosomal position effects when using random insertion based transgenic approaches, we have adopted a PhiC31 integrase mediated cassette exchange method for the efficient insertion of transgenes at single copy within a defined and well characterized chromosomal position, ROSA26. This has enabled the direct comparison of constructs from within the same genomic context and allows a systematic and quantitative assessment of the strengths of the promoters in comparison with the endogenous ROSA26 promoter. The behavior of these exogenous promoters, when integrated at ROSA26 in both sense and antisense orientations, reveals a large variation in their levels of activity. In addition, a subset of promoters, EF1α, UbC and CAG, show an increased activity in the sense orientation as a consequence of integration. Transient transfection experiments confirmed these observations to reflect integration dependent effects and also revealed significant differences in the behaviour of these promoters when delivered transiently or stably. As well as providing an important reference which will facilitate the choice of an appropriate promoter to achieve the desired level of expression for a specific research question, this study also demonstrates the suitability of the cassette exchange methodology for the robust and reliable expression of multiple variant transgenes in ES cells.

RAGE and LRP1 modulation in TgCRND8 mice under condition of B-vitamin deficiency

retromer complex. Methods: We have generated SORLA variants lacking binding sites for GGA, PACS1 and the retromer subunit VPS35, and we have explored the consequence of disrupted adaptor interaction for intracellular transport of the receptor and for APP processing both in cultured cells and in mouse models in vivo.Results:When tested in cultured cells, expression of SORLA variants lacking the GGA binding site caused faulty recycling of the receptor and of APP from the early endosomes back to the cell surface and an increase in soluble APP alpha and a decrease in Abeta production. Disrupting the PACS1 binding site, on the other hand, resulted in shunt of the receptor and of APP to the plasma membrane and in massive increase in Abeta levels. To confirm the relevance of altered SORLA trafficking for APP processing in vivo, we generated ROSA26 knock-in mouse models expressing receptor variants lacking the binding sites for GGA, PACS1, and VPS35. Multiple transgenic lines expressing SORLA trafficking mutants under control of the endogenous ROSA26 promoter or the CAG promoter were produced. Robust expression of the receptor variants was seen in multiple tissues including the brain. Currently, these mouse lines are studied to explore the consequences of altered transport of SORLA for amyloidogenic processes. Conclusions: Our cell data indicated that aberrant targeting of SORLA to the recycling compartment or the plasma membrane causes faulty APP trafficking and imbalance in non-amyloidogenic and amyloidogenic processing fates. Thus, novel mouse models expressing trafficking mutants of SORLA represent unique tools to further validate the relevance of SORLA and adaptor interaction for amyloidogenic processing and neurodegeneration in vivo.

Filamins Regulate Differentiation of Megakaryocytes and Platelets From Cultured Embryonic Stem (ES) Cells.

AbstractAbstract 1618Filamins (Flns) A and B are major non-muscle actin binding proteins that play important roles in cross-linking cortical actin filaments into three-dimensional networks. In addition to their role as cytoskeletal scaffolding molecules, filamins are also known to bind more than 30 other proteins, regulating their subcellular location and coordinating their ability to signal. The role of filamins in hematopoietic stem cell differentiation, however, remains unclear, in part because gene-targeted mice lacking filamins die early on in embryonic development. To investigate the role of filamins A and B in the differentiation of embryonic stem cells (ESCs) along the megakaryocyte/platelet axis, we designed shRNA-containing vectors that targeted both FlnA and B under the control of either the CMV immediate-early promoter (CMV-FlnABLow), or an endogenous Rosa26 promoter (Rosa26-FlnABLow). Compared with wild-type ESCs, FlnABLow ESCs formed small, tightly packed undifferentiated colonies that expressed high levels of the ESC transcription factor, Nanog, and low levels of ERK activity – all indicators of an undifferentiated state. Embryoid prepared from FlnABLow ESCs, were allowed to differentiate, and examined for markers of mesoderm differentiation (Flk-1) and megakaryocyte differentiation (CD41). Whereas Day 6 EB-derived FlnABnormal wild-type cells were 8% Flk-1 positive and 13% CD41 positive, Day 6 FlnABLow cells were 9% Flk-1 positive and only 4% CD41 positive, consistent with the notion that loss of Fln A and B results in a delay of mesoderm to hematopoietic differentiation. To evaluate the effect of Fln knockdown on the ability of the CD41-positive cells to further differentiate into megakaryocytes, form proplatelet extensions, and produce platelets, CD41 positive cells isolated from day 8 EBs were cultured in the presence of a thrombopoietin (TPO)-producing TERT stromal cell line. We found that FlnABLow CD41-positive cells formed far fewer and smaller megakaryocytes compared with their FlnABnormal wild-type counterparts. Proplatelets derived from FlnABLow cells exhibited an abnormal, enlarged morphology with swellings and thick shafts that released platelets prematurely, yielding platelets that were nearly twice the size of those derived from FlnABnormal cells. Taken together, we conclude that not only do filamins function prominently in hematopoietic cell differentiation, they also play an important role in platelet production, likely via their ability to by recruit and organize the necessary signaling molecules near the inner face of the plasma membrane. Disclosures:Newman:New York Blood Center: Membership on an entity's Board of Directors or advisory committees; Children's Hospital of Boston: Membership on an entity's Board of Directors or advisory committees.

Tetracycline-controlled transgene activation using the ROSA26-iM2-GFP knock-in mouse strain permits GFP monitoring of DOX-regulated transgene-expression

BackgroundConditional gene activation is an efficient strategy for studying gene function in genetically modified animals. Among the presently available gene switches, the tetracycline-regulated system has attracted considerable interest because of its unique potential for reversible and adjustable gene regulation.ResultsTo investigate whether the ubiquitously expressed Gt(ROSA)26Sor locus enables uniform DOX-controlled gene expression, we inserted the improved tetracycline-regulated transcription activator iM2 together with an iM2 dependent GFP gene into the Gt(ROSA)26Sor locus, using gene targeting to generate ROSA26-iM2-GFP (R26t1Δ) mice. Despite the presence of ROSA26 promoter driven iM2, R26t1Δ mice showed very sparse DOX-activated expression of different iM2-responsive reporter genes in the brain, mosaic expression in peripheral tissues and more prominent expression in erythroid, myeloid and lymphoid lineages, in hematopoietic stem and progenitor cells and in olfactory neurons.ConclusionsThe finding that gene regulation by the DOX-activated transcriptional factor iM2 in the Gt(ROSA)26Sor locus has its limitations is of importance for future experimental strategies involving transgene activation from the endogenous ROSA26 promoter. Furthermore, our ROSA26-iM2 knock-in mouse model (R26t1Δ) represents a useful tool for implementing gene function in vivo especially under circumstances requiring the side-by-side comparison of gene manipulated and wild type cells. Since the ROSA26-iM2 mouse allows mosaic gene activation in peripheral tissues and haematopoietic cells, this model will be very useful for uncovering previously unknown or unsuspected phenotypes.

Abstract 3250: Bioluminescence imaging of prostate cancer progression and response to therapy in PTEN-deficient mice

Abstract Prostate specific deletion of PTEN function in mice has been reported to recapitulate histological progression of human prostate cancers. Here we describe an improvement on this previously characterized model of prostate cancer by incorporation of a luciferase reporter allele. Specifically, the cancer initiating event is coupled to activation of firefly luciferase from the endogenous ROSA26 promoter in the same prostate epithelial cells. Additionally, we have extensively backcrossed our model onto the albino C57BL/6J background which enhances BLI sensitivity. Consistent with previous studies, we show that progression of prostate cancer from 3-12 weeks is rapid and is correlated with an increase in bioluminescence. However, longitudinal BLI from 12-40 weeks reveals a plateau in bioluminescence signal that is correlated with a halt in cellular proliferation as evidenced by Ki67 staining at necropsy. This may be related to activation of a cellular senescence pathway as reported previously in PTEN deficient mice. We further demonstrate the utility of this model to evaluate the effects of diet induced obesity on disease progression and to monitor response to therapy. Our model may offer advantages in sensitivity and specificity of cancer detection compared to other similar approaches. This work is supported by NIH grant R21 CA137490 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3250.

Generalized tetracycline induced Cre recombinase expression through the ROSA26 locus of recombinant mice

Inducible Cre recombinase systems have been developed to bypass initial lethal phenotypes and to provide access to later embryonic or adult phenotypes. Here we describe the generation of a recombinant mouse that combines a tetracycline dependent switch with generalized Cre recombinase expression by targeting the ubiquitously expressed ROSA26 locus. This transgenic strain was developed using a simplified gene delivery system integrating both elements, the reverse tetracycline controlled trans-activator (rtTA) and rtTA inducible promoter into a single vector. In this transgenic strain, the endogenous ROSA26 promoter drives rtTA expression through a splice acceptor site. The tetracycline inducible promoter, cloned in opposite orientation to the ROSA26 locus and separated from the rtTA element by a 5kb human p53 intron, drives Cre recombinase expression. Crossing these mice with a Cre reporter strain showed that Cre DNA-mediated recombination was ubiquitously and effectively induced during various prenatal developmental windows. Background Cre recombinase expression levels were observed in some tissues in the absence of the inducer, mostly during late embryonic developmental stages and in adult animals. Background recombination levels were low during development and most prominent in nervous tissue. Cre recombinase expression could not be effectively induced in adult animals. While rtTA mRNA levels were high in developmental and adult tissues, Cre recombinase mRNA levels remained low after doxycycline treatment. The mouse strain described here provides a valuable tool to further analyze the function of genes during specific developmental windows, by allowing the effective inactivation of their function throughout defined stages of embryonic development.

Synovial Sarcoma: From Genetics to Genetic-based Animal Modeling

Synovial sarcomas are highly aggressive mesenchymal cancers that show modest response to conventional cytotoxic chemotherapy, suggesting a definite need for improved biotargeted agents. Progress has been hampered by the lack of insight into pathogenesis of this deadly disease. The presence of a specific diagnostic t(X;18) translocation leading to expression of the unique SYT-SSX fusion protein in effectively all cases of synovial sarcoma suggests a role in the etiology. Other nonspecific anomalies such as overexpression of Bcl-2, HER-2/neu, and EGFR have been reported, but their role in the pathogenesis remains unclear. Using gene targeting, we recently generated mice conditionally expressing the human SYT-SSX2 fusion gene from mouse endogenous ROSA26 promoter in chosen tissue types in the presence of Cre recombinase. These mice develop synovial sarcoma when SYT-SSX2 is expressed within myoblasts, thereby identifying a source of this enigmatic tumor and establishing a mouse model of this disease that recapitulates the clinical, histologic, immunohistochemical, and transcriptional profile of human synovial sarcomas. We review the genetics of synovial sarcoma and discuss the usefulness of genetics-based mouse models as a valuable research tool in the hunt for key molecular determinants of this lethal disease as well as a preclinical platform for designing and evaluating novel treatment strategies.

Expression of Transgenes Targeted to the Gt(ROSA)26Sor Locus Is Orientation Dependent

BackgroundTargeting transgenes to a chosen location in the genome has a number of advantages. A single copy of the DNA construct can be inserted by targeting into regions of chromatin that allow the desired developmental and tissue-specific expression of the transgene.MethodologyIn order to develop a reliable system for reproducibly expressing trangenes it was decided to insert constructs at the Gt(ROSA)26Sor locus. A cytomegalovirus (CMV) promoter was used to drive expression of the Tetracycline (tet) transcriptional activator, rtTA2s-M2, and test the effectiveness of using the ROSA26 locus to allow transgene expression. The tet operator construct was inserted into one allele of ROSA26 and a tet responder construct controlling expression of EGFP was inserted into the other allele.ConclusionsExpression of the targeted transgenes was shown to be affected by both the presence of selectable marker cassettes and by the orientation of the transgenes with respect to the endogenous ROSA26 promoter. These results suggest that transcriptional interference from the endogenous gene promoter or from promoters in the selectable marker cassettes may be affecting transgene expression at the locus. Additionally we have been able to determine the optimal orientation for transgene expression at the ROSA26 locus.