Targeting CreERT2 expression to keratin 8-expressing murine simple epithelia using bacterial artificial chromosome transgenesis

Keratin 8 (K8) is a type II keratin that is associated with the type I keratins K18 or K19 in single layered epithelia. We generated a bacterial artificial chromosome (BAC) transgenic mouse line that expresses the tamoxifen inducible CreER(T2) inserted into the endogenous murine K8 gene. The transgenic mouse line contains two copies of the BAC transgene. To determine the expression specificity and inducibility of CreER(T2), the K8-CreER(T2) mice were bred with a Gt(ROSA 26)( ACTB-tdTomato-EGFP ) fluorescent protein-based reporter transgenic mouse line. We demonstrated that CreER(T2) and the endogenous K8 gene share the same patterns of expression and that the enzymatic activity of CreER(T2) can be efficiently induced by tamoxifen in all K8-expressing tissues. This mouse line will be useful for studying gene function in development and homeostasis of simple epithelia, and investigating both tissue lineage hierarchy and the identity of the cells of origin for epithelial cancers.     

Efficient temporally-controlled targeted mutagenesis in smooth muscle cells of the adult mouse

To generate temporally-controlled targeted somatic mutations selectively and efficiently in smooth muscles, we have established a transgenic SMA-Cre-ER(T2) mouse line in which the expression of the Tamoxifen-dependent Cre-ER(T2) recombinase is under the control of a large genomic DNA segment of the mouse smooth muscle alpha actin (SMA) gene, contained in a Bacterial artificial chromosome (Bac). In this transgenic mouse line, Cre-ER(T2)-mediated recombination of LoxP-flanked target DNA is strictly Tamoxifen-dependent, and efficient in both vascular and visceral smooth muscle cells. Moreover, with the exception of few cardiomyocytes, LoxP-flanked DNA excision is restricted to smooth muscle cells. Thus, SMA-Cre-ER(T2) mice should be of great value to analyze gene function in smooth muscles, and to establish new animal models of human smooth muscle disorders.     

Smooth muscle protein 22 alpha-Cre is expressed in myeloid cells in mice

Background: Experiments using Cre recombinase to study smooth muscle specific functions rely on strict specificity of Cre transgene expression. Therefore, accurate determination of Cre activity is critical to the interpretation of experiments using smooth muscle specific Cre. Methods and results: Two lines of smooth muscle protein 22 α-Cre (SM22α-Cre) mice were bred to floxed mice in order to define Cre transgene expression. Southern blotting demonstrated that SM22α-Cre was expressed not only in tissues abundant of smooth muscle, but also in spleen, which consists largely of immune cells including myeloid and lymphoid cells. PCR detected SM22α-Cre expression in peripheral blood and peritoneal macrophages. Analysis of SM22α-Cre mice crossed with a recombination detector GFP mouse revealed GFP expression, and hence recombination, in circulating neutrophils and monocytes by flow cytometry. Conclusions: SM22α-Cre mediates recombination not only in smooth muscle cells, but also in myeloid cells including neutrophils, monocytes, and macrophages. Given the known contributions of myeloid cells to cardiovascular phenotypes, caution should be taken when interpreting data using SM22α-Cre mice to investigate smooth muscle specific functions. Strategies such as bone marrow transplantation may be necessary when SM22α-Cre is used to differentiate the contribution of smooth muscle cells versus myeloid cells to observed phenotypes.     

Characterization of melanocyte-specific inducible Cre recombinase transgenic mice

Conditional Cre-mediated recombination has emerged as a robust method of introducing somatic genetic alterations in an organ-specific manner in the mouse. Here, we generated and characterized mice harboring a 4-hydroxytamoxifen (OHT)-inducible Cre recombinase-estrogen receptor fusion transgene under the control of the melanocyte-specific tyrosinase promoter, designated Tyr::CreER(T2). Cre-mediated recombination was induced in melanocytes in a spatially and temporally controlled manner upon administration of OHT and was documented in embryonic melanoblasts, follicular bulb melanocytes, dermal dendritic melanocytes, epidermal melanocytes of tail skin, and in putative melanocyte stem cells located within the follicular bulge. Functional evidence suggestive of recombination in follicular melanocyte stem cells included the presence of Cre-mediated recombination in follicular bulb melanocytes 1 year after topical OHT administration, by which time several hair cycles have elapsed and the melanocytes residing in this location have undergone multiple rounds of apoptosis and replenishment. These Tyr:: CreER(T2) transgenic mice represent a useful resource for the evaluation of melanocyte developmental genetics, the characterization of melanocyte stem cell function and dynamics, and the construction of refined mouse models of malignant melanoma.     

Temporal regulation of Cre-recombinase activity in Scl-positive neurons of the central nervous system

The Cre/LoxP system provides a powerful tool to investigate gene function in vivo. This system requires Cre-recombinase expressing mouse lines that permit control of gene recombination in a tissue-specific and time-dependent manner. To allow spatio-temporal gene deletion in specific central nervous system (CNS) neuronal populations, we generated mice with a tamoxifen-inducible Cre (Cre-ER(T)) transgene under control of the Scl/Tal1 neural promoter/enhancer -0.9E3 (-0.9E3CreER(T) transgenic mice). Using Cre-reporter mice we have shown that tamoxifen-mediated Cre-ER(T) recombination in -0.9E3CreER(T) mice recapitulated the anticipated expression pattern of Scl in the caudal thalamus, midbrain, hindbrain, and spinal cord. Cre-mediated recombination was also effectively induced during embryogenesis and marked the same population of neurons as observed in the adult. Additionally, we identified a tamoxifen-independent constitutively active -0.9E3CreER(T) mouse line that will be useful for gene deletion during early neurogenesis. These -0.9E3CreER(T) mice will provide tools to investigate the role of neuronal genes in the developing and mature CNS. CNS.     

Local Cre-Mediated Gene Recombination in Vascular Smooth Muscle Cells in Mice

Here we describe a means to conditionally modify genes at a predefined and localized region of the vasculature using a perivascular drug delivery device (PDD). A 4-hydroxytamoxifen (4-OHT)-eluting PDD was applied around the carotid or femoral artery of a mouse strain carrying both the tamoxifen-inducible and smooth muscle cell (SMC)-specific Cre-recombinase (SM-Cre-ER^T2) transgene and a stop-floxed β-galactosidase gene in the Rosa26 locus: the SM-CreER^T2(ki)/rosa26 mouse. A dose and time curve of 0–10% (w/w) 4-OHT and 0–14 days application of the PDD in SM-CreER^T2(ki)/rosa26 mice showed optimal gene recombination at 1% (w/w) 4-OHT loading at 7 days post application (carotid artery 2.4±1.8%; femoral artery 4.0±3.8% of SMCs). The unique 4-OHT-eluting PDD allowed us to achieve SMC-specific recombination in the same order of magnitude as compared to systemic tamoxifen administration. In addition, recombination was completely confined to the PDD-treated vessel wall segment. Thus, local application of a 4-OHT-eluting PDD results in vascular SMC-specific Cre-mediated recombination in SM-CreER^T2(ki)/rosa26 mice without affecting additional SMCs. These authors contributed equally     

Inducible Cre recombinase activity in mouse mature astrocytes and adult neural precursor cells

Two transgenic mouse lines expressing an inducible form of the Cre recombinase (CreER(TM)) under the control of the human GFAP promoter have been generated and characterized. In adult mice, expression of the fusion protein is largely confined to astrocytes in all regions of the central nervous system. Minimal spontaneous Cre activity was detected and recombination was efficiently induced by intraperitoneal administration of tamoxifen in adult mice. The pattern of recombination closely mirrored that of transgene expression. The percentage of astrocytes undergoing recombination varied from region to region ranging from 35% to 70% while a much smaller portion (<1%) of oligodendrocytes and neural precursor cells showed evidence of Cre activity. These mouse lines will provide important tools to dissect gene function in glial cells and in gliomagenesis.     

Pitx3-CreER mice showing restricted Cre expression in developing ocular lens and skeletal muscles

To generate temporally controlled inactivation or activation of interested genes in Pitx3-expressing cells, the tamoxifen-inducible form of Cre, CreER(T2), was inserted into the Pitx3 locus of a mouse BAC clone. Following a single dose of tamoxifen, Cre activity in Pitx3-CreER(T2) transgenic mice was observed in the ocular lens and skeletal muscles but not in the central nervous system at various embryonic stages. This mouse line provides a reagent for driving inducible Cre-dependent recombination in the lens and skeletal muscles during embryonic development.     

A Cre recombinase transgene with mosaic, widespread tamoxifen-inducible action

Cre-mediated site-specific recombination allows conditional transgene expression or gene knockouts in mice. Inducible Cre recombination systems have been developed to bypass initial embryonic lethal phenotypes and provide access to later embryonic or adult phenotypes. We have produced Cre transgenic mice in which excision is tamoxifen inducible and occurs in a widespread mosaic pattern. We utilized our Cre excision reporter system combined with an embryonic stem (ES) cell screen to identify ES cell clones with undetectable background Cre activity in the absence of tamoxifen but efficient excision upon addition of tamoxifen. The CreER transgenic mouse lines derived from the ES cells were tested using the Z/AP and Z/EG Cre reporter lines. Reporter gene expression indicated Cre excision was maximal in midgestation embryos by 2 days after tamoxifen administration, with an overall efficiency of 5-10% of cells with Cre excision. At 3 days after tamoxifen treatment most reporter gene expression marked groups of cells, suggesting an expansion of cells with Cre excision, and the proportion of cells with Cre excision was maintained. In adults, Cre excision was also observed with varying efficiencies in all tissues after tamoxifen treatment.     

Site- and time-specific gene targeting in the mouse

The efficient introduction of somatic mutations in a given gene, at a given time, in a specific cell type, will facilitate studies of gene function and the generation of animal models for human diseases. We have established a conditional site-specific recombination system in mice using a new version of the Cre/lox system. The Cre recombinase has been fused to a mutated ligand binding domain of the human estrogen receptor (ER), resulting in a tamoxifen-dependent Cre recombinase, Cre-ER(T), that is activated by tamoxifen, but not by estradiol. Transgenic mice were generated expressing Cre-ER(T) under the control of a cytomegalovirus promoter. Administration of tamoxifen to these transgenic mice induced excision of a chromosomally integrated gene flanked by loxP sites in a number of tissues, whereas no excision could be detected in untreated animals. However, the efficiency of excision varied between tissues, and the highest level (approximately 40%) was obtained in the skin. To determine the efficiency of excision mediated by Cre-ER(T) in a given cell type, Cre-ER(T)-expressing mice were crossed with reporter mice in which expression of Escherichia coli beta-galactosidase can be induced through Cre-mediated recombination. The efficiency and kinetics of this recombination were analyzed at the cellular level in the epidermis of 6- to 8-week-old double transgenic mice. Site-specific excision occurred within a few days of tamoxifen treatment in essentially all epidermis cells expressing Cre-ER(T). These results indicate that cell-specific expression of Cre-ER(T) in transgenic mice can be used for efficient tamoxifen-dependent Cre-mediated recombination at loci containing loxP sites, to generate site-specific somatic mutations in a spatiotemporally controlled manner. This conditional site-specific recombination system should allow the analysis of knockout phenotypes that cannot be addressed by conventional gene targeting.     

Tamoxifen‐inducible podocyte‐specific iCre recombinase transgenic mouse provides a simple approach for modulation of podocytes in vivo

We report the generation and initial characterization of a mouse line expressing tamoxifen‐inducible improved Cre (iCre) recombinase (iCre‐ER^T2) under the regulation of NPHS2 (podocin) gene promoter. The resulting transgenic mouse line was named podocin‐iCreER^T2 mice. The efficiency of iCre activity was confirmed by crossing podocin‐iCreER^T2 with the ROSA26 reporter mouse. By using the floxed ROSA reporter mice, we found that tamoxifen specifically induced recombination in the kidneys. In the absence of tamoxifen, recombination was undetectable in podocin‐iCreER^T2;ROSA26 mice. However, following intraperitoneal injection of tamoxifen, selective recombination was observed in the podocytes of adult animals. We further examined the efficiency of recombination by assessing various tamoxifen exposure regimens in adult mice. These results suggest that podocin‐iCre‐ER^T2 mouse provides an excellent genetic tool to examine the function of candidate genes in podocytes in a spatially and temporally‐restricted manner. genesis 48:446–451, 2010. © 2010 Wiley‐Liss, Inc.     

Inducible renal principal cell-specific mineralocorticoid receptor gene inactivation in mice

To investigate the role of the mineralocorticoid receptor (MR) in renal ENaC-mediated sodium reabsorption, we have previously used the Cre-loxP system to generate mice with principal-cell specific MR ablation (MR(AQP2Cre) mice). To restrict Cre expression to principal cells, we have used the regulatory elements of the mouse aquaporin-2 (AQP2) gene to drive Cre expression. Since AQP2 is already expressed during renal development, MR ablation took place long before the analysis performed at the adult stage. To investigate whether the early onset of MR ablation affected the adult renal sodium handling, we developed a transgene expressing the CreER(T2) fusion protein under control of the regulatory elements of the AQP2 gene (AQP2CreER(T2)). Immunofluorescence revealed MR loss in the collecting duct (CD) and late connecting tubule after induction of MR ablation by tamoxifen in MR(AQP2CreERT2) mice that equals the MR loss in MR(AQP2Cre) mice. Surprisingly, tamoxifen-independent MR loss is observed in CDs of noninduced mutants without affecting circulating aldosterone levels. Under a low-salt diet, the induced ablation of MR at the adult stage recapitulates the renal sodium wasting observed in mice with constitutive early-onset MR ablation. The AQP2CreER(T2) transgene is a new tool for investigating in vivo the function of genes downstream of MR in renal ENaC-mediated sodium reabsorption by inducible somatic gene inactivation.     

Characterization of Pdgfrb-Cre transgenic mice reveals reduction of ROSA26 reporter activity in remodeling arteries

With the intention to modulate gene expression in vascular mural cells of remodeling vessels, we generated and characterized transgenic mouse lines with Cre recombinase under the control of the platelet-derived growth factor receptor-β promoter, referred to as Tg(Pdgfrb-Cre)(35Vli) . Transgenic mice were crossed with the Gt(ROSA)26Sor(tm1Sor) strain and examined for Cre activation by β-galactosidase activity, which was compared with endogenous Pdgfrb expression. In addition, Pdgfrb-Cre mice were used to drive expression of a conditional myc-tagged Cthrc1 transgene. There was good overlap of β-galactosidase activity with endogenous Pdgfrb immunoreactivity. However, dedifferentiation of vascular mural cells induced by carotid artery ligation revealed a dramatic discrepancy between ROSA26 reporter activity and Pdgfrb promoter driven Cre dependent myc-tagged Cthrc1 transgene expression. Our studies demonstrate the capability of the Pdgfrb-Cre mouse to drive conditional transgene expression as a result of prior Cre-mediated recombination in tissues known to express endogenous Pdgfrb. In addition, the study shows that ROSA26 promoter driven reporter mice are not suitable for lineage marking of smooth muscle in remodeling blood vessels.     

A tetracycline‐inducible and skeletal muscle‐specific Cre recombinase transgenic mouse

We have generated a transgenic mouse that expresses Cre recombinase only in skeletal muscle and only following tetracycline treatment. This spatiotemporal specificity is achieved using two transgenes. The first transgene uses the human skeletal actin (HSA) promoter to drive expression of the reverse tetracycline‐controlled transactivator (rtTA). The second transgene uses a tetracycline responsive promoter to drive the expression of Cre recombinase. We monitored transgene expression in these mice by crossing them with ROSA26 loxP‐LacZ reporter mice, which express β‐galactosidase when activated by Cre. We find that the expression of this transgene is only detectable within skeletal muscle and that Cre expression in the absence of tetracycline is negligible. Cre is readily induced in this model with tetracycline analogs at a range of embryonic and postnatal ages and in a pattern consistent with other HSA transgenic mice. This mouse improves upon existing transgenic mice in which skeletal muscle Cre is expressed throughout development by allowing Cre expression to begin at later developmental stages. This temporal control of transgene expression has several applications, including overcoming embryonic or perinatal lethality due to transgene expression. This mouse is especially suited for studies of steroid hormone action, as it uses tetracycline, rather than tamoxifen, to activate Cre expression. In summary, we find that this transgenic induction system is suitable for studies of gene function in the context of hormonal regulation of skeletal muscle or interactions between muscle and motoneurons in mice. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Targeted somatic mutagenesis in mouse epidermis

Gene targeting in the mouse is a powerful tool to study mammalian gene function. The possibility to efficiently introduce somatic mutations in a given gene, at a chosen time and/or in a given cell type will further improve such studies, and will facilitate the generation of animal models for human diseases. To create targeted somatic mutations in the epidermis, we established transgenic mice expressing the bacteriophage P1 Cre recombinase or the tamoxifen-dependent Cre-ER(T2) recombinase under the control of the human keratin 14 (K14) promoter. We show that LoxP flanked (floxed) DNA segments were efficiently excised in epidermal keratinocytes of K14-Cre transgenic mice. Furthermore, Tamoxifen administration to adult K14-Cre-ER(T2) mice efficiently induced recombination in the basal keratinocytes, whereas no background recombination was detected in the absence of ligand treatment. These two transgenic lines should be very useful to analyse the functional role of a number of genes expressed in keratinocytes.     

Tissue-specific and inducible Cre-mediated recombination in the gut epithelium

We generated two complementary systems for Cre-mediated recombination of target genes in the mouse digestive epithelium and tested them with a Cre-reporter mouse strain. Cre was expressed under the control of a 9 kb regulatory region of the murine villin gene (vil-Cre). Genetic recombination was initiated at embryonic day (E) 9 in the visceral endoderm, and by E12.5 in the entire intestinal epithelium, but not in other tissues. Cre expression was maintained throughout adulthood. Furthermore, transgenic mice bearing a tamoxifen-dependent Cre recombinase (vil-Cre-ERT2) expressed under the control of the villin promoter were created to perform targeted spatiotemporally controlled somatic recombination. After tamoxifen treatment, recombination was detectable throughout the digestive epithelium. The recombined locus persisted for 60 days after tamoxifen administration, despite rapid intestinal cell renewal, indicating that epithelial progenitor cells had been targeted. The villin-Cre and villin-Cre-ERT2 mice provide valuable tools for studies of cell lineage allocation and gene function in the developing and adult intestine.     

Tamoxifen modulates apoptosis in multiple modes of action in CreER mice

Tamoxifen‐inducible Cre (CreER) has become a powerful tool for in vivo manipulation of the genome. Here, we investigated opposing effects of tamoxifen on apoptosis during embryogenesis using Olig2–CreER knock‐in mice, namely, tamoxifen‐induced apoptosis through CreER‐mediated toxicity and cytoprotective activity of tamoxifen independent of CreER. First, we examined tamoxifen‐induced apoptosis; in the homozygous mice, we observed region‐specific apoptosis in the ventral neural tube, with no obvious increase in the heterozygotes. Next, we detected a cytoprotective effect on apoptosis in the homozygous dorsal root ganglia (DRG). This apoptosis is a secondary phenotype of Olig2‐null mice, as Olig2/CreER is not expressed in the DRG. The cytoprotective effect is DRG‐specific, because tamoxifen did not rescue apoptosis in the interdigital mesenchyme. These data indicate that tamoxifen has multiple effects on apoptosis during development and caution that careful examination is necessary when interpreting results obtained from tamoxifen‐induced recombination: in Olig2‐CreER mice, heterozygotes are usable for lineage‐tracing experiment without obvious toxicity, while homozygotes show efficient recombination, despite enhanced apoptosis. genesis 46:775–781, 2008. © 2008 Wiley‐Liss, Inc.     

Spatiotemporal gene control by the Cre-ERT2 system in melanocytes

The organ-specific and temporal control of gene activation/inactivation is a key issue in the understanding of protein function during normal and pathological development and during oncogenesis. We generated transgenic mice bearing a tamoxifen-dependent Cre recombinase (Tyr::Cre-ERT2) gene expressed under the control of a 6.1 kb murine tyrosinase promoter in order to facilitate targeted spatiotemporally controlled somatic recombination in melanoblasts/melanocytes. Cre-ERT2 production was detected in tissues containing melanocytes. After tamoxifen induction at various times during embryogenesis and adulthood in a Cre-responsive reporter mouse strain, genetic recombination was detected in the melanoblasts and melanocytes of the skin. Thus, the Tyr::Cre-ERT2 transgenic mice provides a valuable tool for following this cell lineage and for investigating gene function in melanocyte development and transformation.