Conditional transgene expression mediated by the mouse beta-actin locus

Transgenic mice are an effective model to study gene function in vivo; however, position effects can complicate tissue-specific transgene analysis. To facilitate precise targeting of a transgenic construct into the mouse genome, we combined the Cre/lox and Flp/FRT recombination systems to allow for rapid transgene replacement and conditional transgene expression from the endogenous beta-actin locus. Flp/FRT recombination was used to rapidly exchange FRT-flanked transgene cassettes by recombinase-mediated cassette exchange in embryonic stem cells, while transgene expression can be activated in mice after Cre-mediated excision of a floxed STOP cassette. To validate our system, we analyzed the expression profile of an EGFP reporter gene after integration into the beta-actin locus and Cre-mediated excision of the floxed STOP cassette. Breeding of EGFP reporter mice with various Cre mouse lines resulted in the expected expression profiles, demonstrating the feasibility of the model to facilitate predictable and strong transgene expression in a spatially and temporally controlled manner.     

Sustained mammary gland-directed, ponasterone A-inducible expression in transgenic mice.

The ability to regulate temporal- and spatial-specific expression of target genes in transgenic mice will facilitate analysis of gene function and enable the generation of murine models of human diseases. The genetic analysis of mammary gland tumorigenesis requires the development of mammary gland-specific transgenics, which are tightly regulated throughout the adult mammary epithelium. Analysis of genes implicated in mammary gland tumorigenesis has been hampered by mosaic transgene expression and the findings that homozygous deletion of several candidate genes (cyclin D1, Stat5A, prolactin receptor) abrogates normal mammary gland development. We describe the development of transgenic mouse lines in which sustained transgene expression was inducibly regulated, both specifically and homogeneously, in the adult mammary gland epithelium. Transgenes encoding RXRalpha and a chimeric ecdysone receptor under control of a modified MMTV-LTR, which targets mammary gland expression, were used. These transgenic 'receptor' lines were crossed with transgenic 'enhancer' lines in which the ecdysone/RXR binding site induced ligand-dependent expression of transgenic beta-galactosidase. Pharmacokinetic analysis of a highly bioactive ligand (ponasterone A), identified through screening ecdysteroids from local plants, demonstrated sustained release and transgene expression in vivo. This transgenic model with both tightly regulated and homogeneous transgene expression, which was sustained in vivo using ligands readily extracted from local flora, has broad practical applicability for genetic analysis of mammary gland disease.     

Roles of TGFbeta signaling in epidermal/appendage development

The transforming growth factor beta (TGFbeta) superfamily encompasses a number of structurally related proteins that can be divided into several subfamilies including TGFbetas, activins/inhibins and bone morphogenetic proteins (BMPs). The Smads are major intracellular mediators in transducing the signals of TGFbeta superfamily members, and are abundantly expressed in the developing epidermis and epidermal appendages. Moreover, the phenotypes of transgenic/knockout mice with altered components of the TGFbeta superfamily signaling pathway suggest that TGFbeta superfamily signaling is required for epidermal/appendage development. TGFbeta superfamily members are involved in most events during epidermal/appendage development through the TGFbeta signal transduction pathway and through cross talk with other signaling pathways. Future studies will be instrumental in defining the precise roles for TGFbeta superfamily signaling in epidermal/appendage development.     

Generation of a mouse with conditionally activated signaling through the BMP receptor, ALK2

BMP signaling plays pleiotropic roles in various tissues. Transgenic mouse lines that overexpress BMP signaling in a tissue-specific manner would be beneficial; however, production of each tissue-specific transgenic mouse line is labor-intensive. Here, using a Cre-loxP system, we generated a conditionally overexpressing mouse line for BMP signaling through the type I receptor ALK2 (alternatively known as AVCRI, ActRI, or ActRIA). By mating this line with Cre-expression mouse lines, Cre-mediated recombination removes an intervening floxed lacZ expression cassette and thereby permits the expression of a constitutively active form of Alk2 (caAlk2) driven by a ubiquitous promoter, CAG. Tissue specificity of Cre recombination was monitored by a bicistronically expressed EGFP following Alk2 cDNA. Increased BMP signaling was confirmed by ectopic phosphorylation of SMAD1/5/8 in the areas where Cre recombination had occurred. The conditional overexpression system described here provides versatility in investigating gene functions in a tissue-specific manner without having to generate independent tissue-specific transgenic lines.     

Dynamic expression of a LEF‐EGFP Wnt reporter in mouse development and cancer

We have characterized a transgenic mouse line in which enhanced green fluorescent protein (EGFP) is expressed under the control of multimerized LEF‐1 responsive elements. In embryos, EGFP was detected in known sites of Wnt activation, including the primitive streak, mesoderm, neural tube, somites, heart, limb buds, mammary placodes, and whisker follicles. In vitro cultured transgenic embryonic fibroblasts upregulated EGFP expression in response to activation of Wnt signaling by GSK3β inhibition. Mammary tumor cell lines derived from female LEF‐EGFP transgenic mice treated with the carcinogen 7, 12‐dimethylbenz[a]anthracene (DMBA) also express EGFP. Thus, this transgenic line is useful for ex vivo and in vitro studies of Wnt signaling in development and cancer. genesis 48:183–194, 2010. © 2010 Wiley‐Liss, Inc.     

Immune responses in a mouse model of vitiligo with spontaneous epidermal de‐ and repigmentation

To generate a mouse model of spontaneous epidermal depigmentation, parental h3TA2 mice, expressing both a human‐derived, tyrosinase‐reactive T‐cell receptor on T cells and the matching HLA‐A2 transgene, were crossed to keratin 14‐promoter driven, stem cell factor transgenic (K14‐SCF) mice with intra‐epidermal melanocytes. In resulting Vitesse mice, spontaneous skin depigmentation precedes symmetrical and sharply demarcated patches of graying hair. Whereas the SCF transgene alone dictates a greater retinoic acid receptor‐related orphan receptor gamma (RORγt)⁺ T‐cell compartment, these cells displayed markedly increased IL‐17 expression within Vitesse mice. Similar to patient skin, regulatory T cells were less abundant compared with K14‐SCF mice, with the exception of gradually appearing patches of repigmenting skin. The subtle repigmentation observed likely reflects resilient melanocytes that coexist with skin‐infiltrating, melanocyte‐reactive T cells. Similar repigmenting lesions were found in a different TCR transgenic model of vitiligo developed on an SCF transgenic background, supporting a role for SCF in repigmentation.     

FISH analysis of 142 EGFP transgene integration sites into the mouse genome

Production of transgenic animals is an important technique for studying various biological processes. However, whether the integration of a particular transgene occurs randomly in the mouse genome has not been determined. Analysis by fluorescence in situ hybridization of the integration sites of the 142 EGFP (a mutant of green fluorescent protein) transgenic lines that we produced showed that the transgenes had become incorporated into every mouse chromosome. A single integration site was observed in 82.4% of the lines. The concomitant integrations of transgene into two different loci were observed in 15 cases (10.6%). In 3 cases, the transgenic founder mice showed chimerism in integration sites (2.1%). Chromosomal translocation was observed in 7 cases (4.9%). Moreover, when we statistically analyzed the transgene integration sites of these mouse lines, they were shown to distribute unevenly throughout the genome. This is the first report to analyze the transgene integration sites by producing more than 100 transgenic mouse lines.     

Ubiquitous and uniform in vivo fluorescence in ROSA26-EGFP BAC transgenic mice

Transplantation studies and cell lineage analyses require the ability to explicitly distinguish morphologically identical cells that have an identifiable marker indicating their origin in vivo. Several reporter mouse strains have been generated for such studies, but pancellular detection of the marker in all tissues has not been achieved. In this report, we describe the generation of transgenic mice that express enhanced green fluorescent protein (EGFP) under control of a 187 kb bacterial artificial chromosome (BAC) containing the murine ROSA26 locus, and show several advantages over existing EGFP reporter lines. It is demonstrated that EGFP is ubiquitously and reproducibly expressed from the murine BAC transgene in all organs and tissues analyzed, including the hematolymphoid compartment. Using this new reporter strain in hematopoietic cell transplantation studies, it is demonstrated that leukocytes in recipients maintain uniform transgene expression and are easily distinguished by flow cytometric analysis of live cells. The results suggest that the ROSA26 BAC is an efficient strategy for expressing complex transgene cassettes in vivo.     

Flp recombinase transgenic mice of C57BL/6 strain for conditional gene targeting

We constructed an expression vector of Flp recombinase modified by adding a nuclear localization signal. Injection of the expression vector into fertilized eggs of the C57BL/6 strain yielded transgenic mouse lines expressing the Flp recombinase transgene in the testis. We crossed the transgenic mice to reporter mice carrying the neomycin phosphotransferase gene flanked by target sites of Flp recombinase. Examination of the deletion of the neomycin phosphotransferase gene in the progeny showed that Flp-mediated recombination took place efficiently in vivo in FLP66 transgenic mouse line. These results suggest that the Flp recombinase system is effective in mice and in combination with the Cre recombinase system extends the potentials of gene manipulation in mice. One of the useful applications of FLP66 transgenic mouse line is the removal of marker genes from mice manipulated for the conditional gene targeting with the Cre/loxP system in the pure C57BL/6 genetic background.     

Cre-mediated recombination in the skin melanocyte lineage

Organ-specific expression of a Cre recombinase allows the analysis of gene function in a particular tissue or cell type. Using a 6.1 kb promoter from the mouse tyrosinase gene, we generated and characterized two lines of transgenic mice that express Cre recombinase in melanoblasts. Utilizing a Cre-responsive reporter mouse strain, genetic recombination was detected in the melanoblasts of the skin from embryonic day 11.5. In addition, Cre-expression was detected in the skin and eyes of mice. Cre transgene activity was occasionally detected in the brain and peripheral nerves but not in other tissues. When Tyr::Cre mice were crossed with mice carrying a homozygous loxP conditional mutation for the insulin-like growth factor receptor gene (Igf1r), Cre-melanoblast-specific recombination pattern was confirmed and no abnormal phenotype was observed. In conclusion, Tyr::Cre transgenic mice provide a valuable tool to follow the cell lineage and to examine gene function in melanocyte development and transformation.     

Fibroblast-specific expression of a kinase-deficient type II transforming growth factor beta (TGFbeta) receptor leads to paradoxical activation of TGFbeta signaling pathways with fibrosis in transgenic mice

To better understand the role of disrupted transforming growth factor beta (TGFbeta) signaling in fibrosis, we have selectively expressed a kinase-deficient human type II TGFbeta receptor (TbetaRIIDeltak) in fibroblasts of transgenic mice, using a lineage-specific expression cassette subcloned from the pro-alpha2(I) collagen gene. Surprisingly, despite previous studies that characterized TbetaRIIDeltak as a dominant negative inhibitor of TGFbeta signaling, adult mice expressing this construct demonstrated TGFbeta overactivity and developed dermal and pulmonary fibrosis. Compared with wild type cells, transgenic fibroblasts proliferated more rapidly, produced more extracellular matrix, and showed increased expression of key markers of TGFbeta activation, including plasminogen activator inhibitor-1, connective tissue growth factor, Smad3, Smad4, and Smad7. Smad2/3 phosphorylation was increased in transgenic fibroblasts. Overall, the gene expression profile of explanted transgenic fibroblasts using cDNA microarrays was very similar to that of littermate wild type cells treated with recombinant TGFbeta1. Despite basal up-regulation of TGFbeta signaling pathways, transgenic fibroblasts were relatively refractory to further stimulation with TGFbeta1. Thus, responsiveness of endogenous genes to TGFbeta was reduced, and TGFbeta-regulated promoter-reporter constructs transiently transfected into transgenic fibroblasts showed little activation by recombinant TGFbeta1. Responsiveness was partially restored by overexpression of wild type type II TGFbeta receptors. Activation of MAPK pathways by recombinant TGFbeta1 appeared to be less perturbed than Smad-dependent signaling. Our results show that expression of TbetaRIIDeltak selectively in fibroblasts leads to paradoxical ligand-dependent activation of downstream signaling pathways and causes skin and lung fibrosis. As well as confirming the potential for nonsignaling receptors to regulate TGFbeta activity, these findings support a direct role for perturbed TGFbeta signaling in fibrosis and provide a novel genetically determined animal model of fibrotic disease.     

Neural expression of alpha-internexin promoter in vitro and in vivo

alpha-Internexin is a 66 kDa neuronal intermediate filament protein found most abundantly in the neurons of the nervous systems during early development. To characterize the function of mouse alpha-internexin promoter, we designed two different expression constructs driven by 0.7 kb or 1.3 kb of mouse alpha-internexin 5'-flanking sequences; one was the enhanced green fluorescent protein (EGFP) reporter for monitoring specific expression in vitro, and the other was the cre for studying the functional DNA recombinase in transgenic mice. After introducing DNA constructs into non-neuronal 3T3 fibroblasts and a neuronal Neuro2A cell line by lipofectamine transfection, we observed that the expression of EGFP with 1.3 kb mouse alpha-internexin promoter was in a neuron-dominant manner. To establish a tissue-specific pattern in the nervous system, we generated a transgenic mouse line expressing Cre DNA recombinase under the control of 1.3 kb alpha-Internexin promoter. The activity of the Cre recombinase at postnatal day 1 was examined by mating the cre transgenic mice to ROSA26 reporter (R26R) mice with knock-in Cre-mediated recombination. Analyses of postnatal day 1 (P1) newborns showed that beta-galactosidase activity was detected in the peripheral nervous system (PNS), such as cranial nerves innervating the tongue and the skin as well as spinal nerves to the body trunk. Furthermore, X-gal-labeled dorsal root ganglionic (DRG) neurons showed positive for alpha-Internexin in cell bodies but negative in their spinal nerves. The motor neurons in the spinal cord did not exhibit any beta-galactosidase activity. Therefore, the cre transgene driven by mouse alpha-internexin promoter, described here, provides a useful animal model to specifically manipulate genes in the developing nervous system.     

A Cre gene directed by a human TSPY promoter is specific for germ cells and neurons

The testis-specific protein Y-encoded (TSPY) gene is a candidate for the gonadoblastoma locus on the Y chromosome and is expressed in normal testicular germ cells and gonadoblastoma cells of XY sex-reversed females. Although TSPY expression has been demonstrated in gonadoblastoma tissues, it is uncertain if such expression is involved in a causative or consequential event of the oncogenic process. We postulate that if TSPY is involved in gonadoblastoma development, its promoter should be functional in the female gonad before and/or at early stages of tumorigenesis. To test this hypothesis, we generated several lines of transgenic mice harboring a Cre-recombinase transgene directed by a 2.4-kb hTSPY promoter. These mice were crossed with the Z/EG reporter line that expresses EGFP only after a Cre-mediated recombination. Our results showed that hTSPY-Cre;Z/EG double transgenic mice expressed EGFP specifically in the germ cells of both male and female gonads. Further, neurons of the central and peripheral nervous systems also expressed EGFP as early as E12.5 embryonic stage. EGFP was particularly observed in the trigeminal nerve, trigeminal ganglion, dorsal root of the ganglia, and in postnatal and adult brains. These observations support the hypothesis that TSPY plays an active role in gonadoblastoma. The tissue-specific expression of the hTSPY-Cre transgene should also be useful in studies utilizing Cre-mediated gene activation/inactivation strategies in gamatogenesis and/or neurogenesis.     

Coat color-tagged green mouse with EGFP expressed from the RNA polymerase II promoter

Laborious molecular genotyping and variegated gene expression are two widely encountered issues for transgenic mouse studies. To facilitate genotyping in the FVB/N albino background and to reduce variegated expression, we successfully generated double-tagged transgenic mice for direct visual genotyping with the coat color phenotype derived from tyrosinase cDNA driven by the tyrosinase promoter and with simultaneous high enhanced green fluorescent protein (EGFP) expression driven by the promoter of RNA polymerase II large subunit gene. Incorporation of insulator into a transgene construct achieved high efficiency of transgene expression in more than 90% of the founders. EGFP was detected as early as the one-cell fertilized egg and lasted for the whole embryo development, as well as in all of the adult tissues examined. The coat color-tagged green mice offer opportunities in applications such as tissue transplantation, lineage tracing, chimera biology, RNA interference, and other transgenic studies.     

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