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  • 1
    Keywords: RECEPTOR ; CELLS ; EXPRESSION ; IN-VITRO ; CELL ; SYSTEM ; TOOL ; GENE ; cell line ; TRANSDUCTION ; INFECTION ; murine ; recombination ; antibodies ; antibody ; TARGET ; virus ; MOUSE ; hormone ; VECTORS ; CELL-LINE ; FUSION ; LINE ; CARCINOMA-CELLS ; MAMMALIAN-CELLS ; HEMATOPOIETIC PROGENITOR CELLS ; RETROVIRAL VECTORS ; VIRAL VECTORS ; PLASMID DNA ; TRANSGENE EXPRESSION ; HIGH-TITER ; gene transfer,ES cells,MESV retroviral vectors,adenoviral vectors,Cre recombinase,Cre.PR fusion ; RECOMBINANT ADENOVIRUS ; SOMATIC MUTAGENESIS
    Abstract: Background Genetic modification of embryonic stem (ES) cells represents a powerful tool for transgenic and developmental experiments. We report that retroviral constructs based on murine embryonal stem cell virus (MESV) can efficiently deliver and express Cre recombinase or a post-translationally inducible Cre-Progesterone receptor (Cre.PR) fusion in mouse fibroblasts and ES cells.Methods To study the vectors a sensitive reporter cell line, 3TZ, was derived from the murine 3T6 fibroblast line that expresses beta-galactosidase only upon Cre-mediated recombination. This was used together with the ROSA26-R ES cell Cre-reporter system or unmodified mouse ES cells as targets of infection. Efficiency of gene transfer was evaluated immunohistochemically by the use of an anti-Cre polyclonal antibody, and by monitoring the expression of beta-galactosidase.Results Infection of the 3TZ cells with high titer 718C or 719CP virus revealed efficient gene transduction of constitutive or hormone-inducible recombinase activity, respectively. The vectors efficiently transduced murine ES cells with Cre, Cre-PR (fusion of Cre and progesterone receptor) or beta-galactosidase. Cre-mediated recombination in more than 60% of ROSA26-R ES cells was achieved when infected by a VSV-G-pseudotyped MESV retrovirus at MOI of 50.Conclusions The MESV-based retroviral systems, when combined with hormone inducible Cre, represent efficient tools for the transfer of Cre activity in ES cells. Copyright (C) 2004 John Wiley Sons, Ltd
    Type of Publication: Journal article published
    PubMed ID: 14716675
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  • 2
    Keywords: brain ; RECEPTOR ; CELLS ; PATHWAY ; PATHWAYS ; GENE ; GENES ; RELEASE ; RESPONSES ; MECHANISM ; FREQUENCY ; hormone ; STRESS ; inactivation ; SIGNALING PATHWAY ; SIGNALING PATHWAYS ; glucocorticoid receptor ; LIVING CELLS ; RECEPTORS ; GLUCOCORTICOID-RECEPTOR ; ANTAGONIST ; rodent ; SUBCELLULAR-LOCALIZATION ; signaling ; NEURONS ; LIFE ; ENHANCEMENT ; ESTROGEN ; corticosteroid ; mineralocorticoid receptor ; LEVEL ; function ; alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor CA1 hippocampus ; glucocorticoid receptor knockout ; MICRODIALYSIS ; mineralocorticoid receptor knockout ; miniature excitatory postsynaptic current ; RAT HIPPOCAMPUS ; SYNAPSES
    Abstract: The adrenal hormone corticosterone transcriptionally regulates responsive genes in the rodent hippocampus through nuclear mineralocorticoid and glucocorticoid receptors. Via this genomic pathway the hormone alters properties of hippocampal cells slowly and for a prolonged period. Here we report that corticosterone also rapidly and reversibly changes hippocampal signaling. Stress levels of the hormone enhance the frequency of miniature excitatory postsynaptic potentials in CA1 pyramidal neurons and reduce paired-pulse facilitation, pointing to a hormone-dependent enhancement of glutamate-release probability. The rapid effect by corticosterone is accomplished through a nongenomic pathway involving membrane-located receptors. Unexpectedly, the rapid effect critically depends on the classical mineralocorticoid receptor, as evidenced by the effectiveness of agonists, antagonists, and brain-specific inactivation of the mineralocorticoid but not the glucocorticoid receptor gene. Rapid actions by corticosterone would allow the brain to change its function within minutes after stress-induced elevations of corticosteroid levels, in addition to responding later through gene-mediated signaling pathways
    Type of Publication: Journal article published
    PubMed ID: 16361444
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  • 3
    Abstract: Lentiviral expression vectors are powerful tools for gene therapy and long-term gene expression/repression in the mammalian brain. However, no specificity of transduction has been reported so far in the central nervous system. Here we have developed a novel system to achieve a neuronal subtype specific expression in either dopaminergic (DA) or GABAergic neurons. We employed a delivery strategy by which the transgene is not expressed until its activation by Cre recombinase. We successfully tested the system in vitro and then used this novel lentivector, containing loxP sites, in 2 different transgenic mouse lines expressing Cre either in DA or in GABAergic neurons. In both lines the reporter gene was detected exclusively in Cre-positive cells, demonstrating that with this experimental approach we were able to achieve completely specific expression of transgenes delivered by lentiviral vectors. This universal system can be applied to all neural subtypes making use of the growing number of specific Cre driver lines.- Tolu, S., Avale, M. E., Nakatani, H., Pons, S., Parnaudeau, S., Tronche, F., Vogt, A., Monyer, H., Vogel, R., de Chaumont, F., Olivo-Marin, J.-C., Changeux, J.-P., Maskos, U. A versatile system for the neuronal subtype specific expression of lentiviral vectors.
    Type of Publication: Journal article published
    PubMed ID: 19858094
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  • 4
    Keywords: APOPTOSIS ; EXPRESSION ; GROWTH-FACTOR ; IN-VIVO ; TUMOR-NECROSIS-FACTOR ; ACTIVATION ; NUCLEAR-FACTOR ; CELL-CYCLE PROGRESSION ; TERMINAL PHOSPHORYLATION ; HEPATIC REGENERATION
    Abstract: Mice lacking the AP-1 transcription factor c-jun die at mid-gestation showing heart defects and impaired hepatogenesis. To inactivate c-jun in hepatocytes, mice carrying a floxed c-jun allele were generated. Perinatal liver-specific c-jun deletion caused reduced hepatocyte proliferation and decreased body size. After partial hepatectomy, half of the mutants died and liver regeneration was impaired. This phenotype was not present in mice lacking the N-terminal phosphorylation sites of c-Jun. The failure to regenerate was accompanied by increased cell death and lipid accumulation in hepatocytes. Moreover, cyclin-dependent kinases and several cell cycle regulators were affected, resulting in inefficient G(1)-S phase progression. These studies identify c-Jun as a critical regulator of hepatocyte proliferation and survival during liver development and regeneration.
    Type of Publication: Journal article published
    PubMed ID: 11927562
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  • 5
    Keywords: EXPRESSION ; SURVIVAL ; Germany ; PATHWAY ; PATHWAYS ; GENERATION ; SYSTEM ; DISEASE ; DISEASES ; GENE ; PROTEIN ; transcription ; MICE ; ACTIVATION ; TRANSCRIPTION FACTOR ; BINDING ; PHOSPHORYLATION ; CREB ; ELEMENT-BINDING PROTEIN ; TRANSCRIPTION FACTORS ; DISRUPTION ; MUTATION ; LINE ; inactivation ; SIGNALING PATHWAY ; SIGNALING PATHWAYS ; PHENOTYPE ; MUTANT MICE ; CYCLIC-AMP ; BINDING PROTEIN ; signaling ; MICE LACKING ; function ; LOSSES ; neuron ; CAMP RESPONSE ELEMENT ; dopaminergic neurons ; MESENCEPHALIC DOPAMINERGIC-NEURONS ; MIDBRAIN ; TRANSPORTER MESSENGER-RNA
    Abstract: cAMP response element binding protein (CREB) and the related factors CREM (cAMP response element modulator) and ATF1 (activation transcription factor 1) are bZIP-domain-containing transcription factors activated through cAMP and other signaling pathways. The disruption of CREB function in developing and mature neurons affects their development and survival when associated with loss of CREM. Since dopaminergic (DA) neurons are affected in several neurological diseases, we generated CREB conditional mutants in DA neurons by using a newly generated transgenic Cre line targeting the dopaminergic system (DATCre). Here we report the generation and analysis of mutant mice lacking CREB in DA neurons (CREBDATCre mutants). During adulthood, lack of CREB leads to a partial loss of DA neurons. Since CREM is upregulated in absence of CREB, we have introduced this mutation in a CREM-/- genetic background to assess a compensatory role of CREM. Additional inactivation of CREM does not lead to a more severe phenotype
    Type of Publication: Journal article published
    PubMed ID: 16981198
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  • 6
    Keywords: RECEPTOR ; EXPRESSION ; NETWORK ; GENE ; GENE-EXPRESSION ; GENES ; MICE ; MESSENGER-RNA ; primary ; MR ; culture ; MOUSE ; IDENTIFICATION ; gene expression ; CELL-DEATH ; genetics ; INVOLVEMENT ; glucocorticoid receptor ; specificity ; heredity ; genomics ; HEART-FAILURE ; mineralocorticoid receptor ; analysis ; USA ; corticosteroids ; GLUCOCORTICOIDS ; genomic ; microbiology ; SET ; transcriptome ; biotechnology ; PERIPHERAL-TISSUES ; aldosterone ; CA2+ CURRENT ; cardiomyocytes ; gene networks ; NEONATAL-RAT ; SODIUM-TRANSPORT
    Abstract: Corticosteroids (aldosterone, cortisol/corticosterone) exert direct functional effects on cardiomyocytes. However, gene networks activated by corticosteroids in cardiomyocytes, as well as the involvement of the mineralocorticoid receptor (MR) vs the glueocorticoid receptor (GR) in these effects, remain largely unknown. Here we characterized the corticosteroid-dependent transcriptome in primary culture of neonatal mouse cardiomyocytes treated with 10(-6) M aldosterone, a concentration predicted to occupy both MR and GR. Serial analysis of gene expression revealed 101 alclosterone-regulated genes. The MR/GR specificity was characterized for one regulated transcript, namely ecto-ADP-ribosyltransferase-3 (Art3). Using cardiomyocytes from GR(null/null) or MR(null/null) mice we demonstrate that in GR(null/null) cardiomyocytes the response is abrogated, but it is fully maintained in MR(null/null) cardiomyocytes. We conclude that Art3 expression is regulated exclusively via the GR. Our study identifies a new set of corticosteroid-regulated genes in cardiomyocytes and demonstrates a new approach to studying the selectivity of MR- vs GR-dependent effects. (c) 2006 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 17174066
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  • 7
    Keywords: RECEPTOR ; CELLS ; CELL ; Germany ; GENE ; PROTEIN ; PROTEINS ; TISSUE ; MICE ; TUMOR-NECROSIS-FACTOR ; DNA ; MACROPHAGES ; MECHANISM ; CONTRAST ; DENDRITIC CELLS ; KERATINOCYTES ; mechanisms ; SKIN ; T cell ; T cells ; T-CELL ; T-CELLS ; SUPPRESSION ; treatment ; cytokines ; TARGET ; MUTANT ; inactivation ; DNA-BINDING ; BETA ; MOUSE MODEL ; TARGETS ; side effects ; REPRESSION ; DIMERIZATION ; chemokine ; TNF-ALPHA ; NEUTROPHILS ; CYTOKINE ; molecular ; PERSISTENT ; RECOMBINANT ; INFILTRATION ; MOLECULAR-MECHANISM ; RE ; keratinocyte ; allergy ; IMMUNE SUPPRESSION ; chemokines ; INFLAMMATORY CYTOKINES ; MOLECULAR-MECHANISMS ; PHASE ; USA ; corticosteroids ; GLUCOCORTICOIDS ; RESISTANT ; SKIN INFLAMMATION ; CONTACT ; MEDICINE ; INFLAMMATORY RESPONSE ; EPIDERMAL LANGERHANS CELLS ; HYPERSENSITIVITY REACTIONS ; INFLAMMATORY PROTEIN-2
    Abstract: Glucocorticoids (GCs) are widely used in the treatment of allergic skin conditions despite having numerous side effects. Here we use Cre/loxP-engineered tissue- and cell-specific and function-selective GC receptor (GR) mutant mice to identify responsive cell types and molecular mechanisms underlying the and inflammatory activity of GCs in contact hypersensitivity (CHS). CHS was repressed by GCs only at the challenge phase, i.e., during reexposure to the hapten. Inactivation of the GR gene in keratinocytes or T cells of mutant mice did not attenuate the effects of GCs, but its ablation in macrophages and neutrophils abolished downregulation of the inflammatory response. Moreover, mice expressing a DNA binding-defective GR were also resistant to GC treatment. The persistent infiltration of macrophages and neutrophils in these mice is explained by an impaired repression of inflammatory cytokines and chemokines such as IL-1 beta, monocyte chemoattractant protein-1, macrophage inflammatory protein-2, and IFN-gamma-inducible protein 10. In contrast TNF-alpha repression remained intact. Consequently, injection of recombinant proteins of these cytokines and chemokines partially reversed suppression of CHS by GCs. These studies provide evidence that in contact allergy, therapeutic action of corticosteroids is in macrophages and neutrophils and that dimerization GR is required
    Type of Publication: Journal article published
    PubMed ID: 17446934
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  • 8
    Keywords: brain ; CELLS ; EXPRESSION ; CELL ; MODEL ; GENERATION ; TOOL ; ENZYMES ; GENE ; GENE-EXPRESSION ; PROTEIN ; PROTEINS ; LINES ; MICE ; DNA ; recombination ; BIOLOGY ; MOLECULAR-BIOLOGY ; chromosome ; NUCLEI ; ELEMENT ; MOUSE ; TRANSGENIC MICE ; DISTRIBUTIONS ; IN-SITU ; immunohistochemistry ; gene expression ; ESCHERICHIA-COLI ; ELEMENTS ; COMPONENT ; LINE ; LOCALIZATION ; LIVING CELLS ; CRE RECOMBINASE ; targeting ; molecular biology ; molecular ; PATTERN ; CRE ; NEURONS ; LEVEL ; ENZYME ; analysis ; ANIMAL-MODEL ; function ; animal ; neuron ; animal model ; - ; DOPAMINERGIC-NEURONS ; PLASTICITY ; ARCUATE NUCLEUS ; ARTIFICIAL CHROMOSOME ; Cre/loxP system ; dopamine transporter ; dopaminergic cells ; NONDOPAMINERGIC NEURONS ; synapse
    Abstract: The dopamine transporter is an essential component of the dopaminergic synapse. It is located in the presynaptic neurons and regulates extracellular dopamine levels. We generated a transgenic mouse line expressing the Cre recombinase under the control of the regulatory elements of the dopamine transporter gene, for investigations of gene function in dopaminergic neurons. The codon-improved Cre recombinase (iCre) gene was inserted into the dopamine transporter gene on a bacterial artificial chromosome. The pattern of expression of the bacterial artificial chromosome-dopamine transporter-iCre transgene was similar to that of the endogenous dopamine transporter gene, as shown by immunohistochemistry. Recombinase activity was further studied in mice carrying both the bacterial artificial chromosome-dopamine transporter-iCre transgene and a construct expressing the beta-galactosidase gene after Cre-mediated recombination. In situ studies showed that beta-galactosidase (5-bromo-4-chloroindol-3-yl beta-D-galactoside staining) and the dopamine transporter (immunofluorescence) had identical distributions in the ventral midbrain. We used this animal model to study the distribution of dopamine transporter gene expression in hypothalamic nuclei in detail. The expression profile of tyrosine hydroxylase (an enzyme required for dopamine synthesis) was broader than that of beta-galactosidase in A12 to A15. Thus, only a fraction of neurons synthesizing dopamine expressed the dopamine transporter gene. The bacterial artificial chromosome-dopamine transporter-iCre transgenic line is a unique tool for targeting Cre/loxP-mediated DNA recombination to dopamine neurons for studies of gene function or for labeling living cells, following the crossing of these mice with transgenic Cre reporter lines producing fluorescent proteins
    Type of Publication: Journal article published
    PubMed ID: 17565601
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  • 9
    Keywords: brain ; EXPRESSION ; transcription ; TRANSGENIC MICE ; BEHAVIOR ; secretion ; CORTICOTROPIN-RELEASING-FACTOR ; IMPAIRED STRESS-RESPONSE ; ELEVATED PLUS-MAZE ; ADRENOCORTICAL REGULATION
    Abstract: The glucocorticoid receptor (Gr, encoded by the gene Grl1) controls transcription of target genes both directly by interaction with DNA regulatory elements and indirectly by cross-talk with other transcription factors. In response to various stimuli, including stress, glucocorticoids coordinate metabolic, endocrine, immune and nervous system responses and ensure an adequate profile of transcription. In the brain, Gr has been proposed to modulate emotional behaviour, cognitive functions and addictive states. Previously, these aspects were not studied in the absence of functional Gr because inactivation of Grl1 in mice causes lethality at birth (F.T., C.K. and G.S., unpublished data). Therefore, we generated tissue-specific mutations of this gene using the Cre/loxP -recombination system. This allowed us to generate viable adult mice with loss of Gr function in selected tissues. Loss of Gr function in the nervous system impairs hypothalamus-pituitary-adrenal (HPA)-axis regulation, resulting in increased glucocorticoid (GC) levels that lead to symptoms reminiscent of those observed in Cushing syndrome. Conditional mutagenesis of Gr in the nervous system provides genetic evidence for the importance of Gr signalling in emotional behaviour because mutant animals show an impaired behavioural response to stress and display reduced anxiety.
    Type of Publication: Journal article published
    PubMed ID: 10471508
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  • 10
    Abstract: The pituitary adenylate cyclase activating polypeptide (PACAP) type I receptor (PAC1) is a G-protein-coupled receptor binding the strongly conserved neuropeptide PACAP with 1000-fold higher affinity than the related peptide vasoactive intestinal peptide. PAC1-mediated signaling has been implicated in neuronal differentiation and synaptic plasticity. To gain further insight into the biological significance of PAC1-mediated signaling in vivo, we generated two different mutant mouse strains, harboring either a complete or a forebrain-specific inactivation of PAC1. Mutants from both strains show a deficit in contextual fear conditioning, a hippocampus-dependent associative learning paradigm. In sharp contrast, amygdala-dependent cued fear conditioning remains intact. Interestingly, no deficits in other hippocampus-dependent tasks modeling declarative learning such as the Morris water maze or the social transmission of food preference are observed. At the cellular level, the deficit in hippocampus-dependent associative learning is accompanied by an impairment of mossy fiber long-term potentiation (LTP). Because the hippocampal expression of PAC1 is restricted to mossy fiber terminals, we conclude that presynaptic PAC1-mediated signaling at the mossy fiber synapse is involved in both LTP and hippocampus-dependent associative learning.
    Type of Publication: Journal article published
    PubMed ID: 11466423
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