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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 ; 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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  • 3
    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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  • 4
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  • 5
    Keywords: brain ; RECEPTOR ; MODEL ; GENE ; transcription ; DRUG ; MICE ; TRANSCRIPTION FACTOR ; TARGET ; NERVOUS-SYSTEM ; c-Fos ; STRESS ; transgenic ; AMPHETAMINE ; cocaine ; CORTICOSTERONE ; DOPAMINERGIC TRANSMISSION ; glucocorticoid receptor ; INDIVIDUAL VULNERABILITY ; INDUCED SENSITIZATION ; intravenous ; mifepristone ; PROGRESSIVE RATIO SCHEDULE ; SEEKING BEHAVIOR ; self-administration ; sensitization
    Abstract: Several findings suggest that glucocorticoid hormones are involved in determining the propensity of an individual to develop cocaine abuse. These hormones activate two related transcription factors, the glucocorticoid receptor (GR) and the mineralocorticoid receptor. In this study, we show that the selective inactivation of the GR gene in the brains of mice profoundly flattened the dose - response function for cocaine intravenous self-administration and suppressed sensitization, two experimental procedures considered relevant models of addiction. Furthermore, administration of a GR antagonist dose- dependently reduced the motivation to self-administer cocaine. Importantly, the absence of GR did not modify the basal behavioral and molecular effects of cocaine but selectively modified the excessive response to the drug spontaneously present in certain vulnerable individuals or induced by repeated drug exposure in others. In conclusion, we provide the first genetic evidence that the GR gene can modulate cocaine abuse. This suggests that targeting GR function in the brain could provide new therapeutic strategies to treat cocaine addiction for which there is no available treatment
    Type of Publication: Journal article published
    PubMed ID: 12805318
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  • 6
    Keywords: RECEPTOR ; EXPRESSION ; BLOOD ; CELL ; Germany ; IN-VIVO ; liver ; ENZYMES ; GENE ; GENES ; transcription ; METABOLISM ; MICE ; ACTIVATION ; kidney ; TRANSCRIPTION FACTOR ; INDUCTION ; hepatocytes ; MUTANT ; hormone ; DISRUPTION ; TANDEM MASS-SPECTROMETRY ; inactivation ; Jun ; GLUCOSE ; glucocorticoid receptor ; GLUCOCORTICOID-RECEPTOR ; ANTAGONIST ; insulin ; ABSENCE ; ADULT ; ENDOCRINE ; LEADS ; development ; CARBOXYKINASE GTP GENE ; HEPATIC GLUCONEOGENESIS ; PHOSPHOENOLPYRUVATE CARBOXYKINASE ; TYROSINE AMINOTRANSFERASE GENE
    Abstract: Hepatic glucose production by gluconeogenesis is the main source of glucose during fasting and contributes significantly to hyperglycemia in diabetes mellitus. Accordingly, glucose metabolism is tightly controlled by a variety of hormones including insulin, epinephrine, glucagon, and glucocorticoids (GCs) acting on various cell types. GC effects are mediated by the GC receptor (GR), a ligand-dependent transcription factor, which in the liver and kidney controls gluconeogenesis by induction of gluconeogenic enzymes. To specifically study the contribution of GC on liver carbohydrate metabolism, we generated mice with an inactivation of the GR gene exclusively in hepatocytes using the Cre/loxP technology. Half of the mutant mice die within the first 2 d after birth most likely due to hypoglycemia. Adult mice have normal blood sugar under basal conditions but show hypoglycemia after prolonged starvation due to reduced expression of genes involved in gluconeogenesis. We further demonstrate that absence of GR in hepatocytes limits the development of hyperglycemia in streptozotocin-induced diabetes mellitus probably due to impaired induction of gluconeogenesis. These findings show the essential role of GR function in liver glucose metabolism during fasting and in diabetic mice and indicate that liver-specific GC antagonists could be beneficial in control of diabetic hyperglycemia
    Type of Publication: Journal article published
    PubMed ID: 15031319
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  • 7
    Keywords: MESSENGER-RNA ; BODY-WEIGHT ; OBESITY ; FOOD-INTAKE ; NEUROPEPTIDE-Y ; CORTICOTROPIN-RELEASING FACTOR ; GLUCOCORTICOID RECEPTOR GENE ; LEPTIN RESPONSIVENESS ; FEEDBACK-CONTROL ; ZUCKER RATS
    Abstract: The homeostatic regulation of body weight protects the organism from the negative consequences of starvation and obesity. Glucocorticoids (GCs) modulate this regulation, although the underlying mechanisms remain unclear. To address the role of central GRs in the regulation of energy balance, we studied mice in which GRs have selectively been inactivated in the nervous system. Mutant mice display marked growth retardation. During suckling age this is associated with normal fat deposition causing a 60% temporary increase of percent body fat, compared with control littermates. After weaning, fat and protein depositions are reduced so that adults are both smaller and leaner than their controls. Decreased food intake and, after weaning, reduced metabolic efficiency account for these developmental disturbances. Plasma levels of leptin and insulin, two important energy balance regulators, are elevated in young mutants but normal in adults. Leptin/body fat ratio is higher at all ages, suggesting disturbed control of circulating leptin as a consequence of chronically elevated GC levels in mutant animals. Adult mutants display increased hypothalamic CRH and NPY levels, but peptide levels of melanin concentrating hormone and Orexin A and B are unchanged. The increased levels of plasma GCs and hypothalamic CRH may act as catabolic signals most likely leading to persistently reduced energy accumulation.
    Type of Publication: Journal article published
    PubMed ID: 12021198
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  • 8
    Keywords: MESSENGER-RNA ; TRANSGENIC MICE ; DNA-BINDING ; GLUCOCORTICOID-RECEPTOR ; mineralocorticoid receptor ; PITUITARY-ADRENOCORTICAL AXIS ; CORTICOTROPIN-RELEASING HORMONE ; IMPAIRED STRESS-RESPONSE ; MEMORY FORMATION ; ANXIETY-LIKE BEHAVIOR
    Abstract: Corticosteroids are released by the adrenal cortex with a diurnal rhythm and in response to stressful environmental changes. They not only act on peripheral organs, but also regulate brain physiology, thereby affecting mental processes like emotion and cognition. Here, we discuss the role of the two known corticosteroid receptors--glucocorticoid receptor (GR) and mineralocorticoid receptor (MR)--in the brain by summarizing the results obtained with various genetically modified mouse lines. In these lines, either the GR or the MR gene has been targeted or GR protein levels have been upregulated or downregulated. Analysis of the different lines confirms the importance of GR in the regulation of the hypothalamic pituitary adrenal (HPA) axis because interference with GR activity activates the HPA axis, whereas increased GR protein levels inhibit HPA axis activity. Genetic downregulation of GR protein levels and inactivation of the GR gene in the brain reduce anxiety-related behavior, which reveals a central role of GR in emotional behavior. Both HPA axis activity and anxiety are modulated by corticotropin releasing hormone (CRH); therefore, we include in the discussion results obtained with genetically modified CRH or CRH receptor mice. We further address the important role of corticosteroid receptors for hippocampal function and integrity. Cellular properties of CA1 neurons are changed, and hippocampal-dependent explicit memory is affected in GR mutant animals. Comparing MR and GR mutant animals suggests the requirement of MR but not GR for dentate gyrus granule cell maintenance. Because an imbalance in glucocorticoid levels is associated with cognitive impairments and mental disorders, the described mouse lines will aid in understanding the mechanisms involved in the pathology of these disorders.
    Type of Publication: Journal article published
    PubMed ID: 11827739
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  • 9
    Keywords: SURVIVAL ; GENE ; ELEMENT-BINDING PROTEIN ; CELL-DEATH ; MUTANT MICE ; BCL-2 EXPRESSION ; MICE LACKING ; HUNTINGTONS-DISEASE ; EXPANDED POLYGLUTAMINE ; MEDIATED TRANSCRIPTION
    Abstract: Control of cellular survival and proliferation is dependent on extracellular signals and is a prerequisite for ordered tissue development and maintenance. Activation of the cAMP responsive element binding protein (CREB) by phosphorylation has been implicated in the survival of mammalian cells. To define its roles in the mouse central nervous system, we disrupted Creb1 in brain of developing and adult mice using the Cre/loxP system. Mice with a Crem(-/-) background and lacking Creb in the central nervous system during development show extensive apoptosis of postmitotic neurons. By contrast, mice in which both Creb1 and Crem are disrupted in the postnatal forebrain show progressive neurodegeneration in the hippocampus and in the dorsolateral striatum. The striatal phenotype is reminiscent of Huntington disease and is consistent with the postulated role of CREB-mediated signaling in polyglutamine-triggered diseases.
    Type of Publication: Journal article published
    PubMed ID: 11967539
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  • 10
    Keywords: RECEPTOR ; GROWTH ; CELL ; Germany ; IN-VIVO ; MODEL ; VIVO ; SUPPORT ; liver ; GENE ; GENE-EXPRESSION ; PROTEIN ; PROTEINS ; transcription ; MICE ; ACTIVATION ; TRANSCRIPTION FACTOR ; REDUCTION ; hepatocytes ; TRANSCRIPTION FACTORS ; hormone ; inactivation ; DNA-BINDING ; REGION ; REGIONS ; TARGETED DISRUPTION ; BINDING PROTEIN-3 ; I IGF-I ; postnatal body growth,glucocorticoid receptor,growth hormone signaling,Stat5 ; STAT5B
    Abstract: Mice carrying a hepatocyte-specific inactivation of the glucorticoid receptor (GR) gene show a dramatic reduction in body size. Growth hormone signaling mediated by the Stat5 transcription factors is impaired. We show that Stat5 proteins physically interact with GR and GR is present in vivo on Stat5-dependent IGF-I and ALS regulatory regions. Interestingly, mice with a DNA-binding-deficient GR but an unaltered ability to interact with STAT5(GR(dim/dim)) have a normal body size and normal levels of Stat5-dependent mRNAs. These findings strongly support the model in which GR acts as a coactivator for Stat5-dependent transcription upon GH stimulation and reveal an essential role of hepatic GR in the control of body growth
    Type of Publication: Journal article published
    PubMed ID: 15037546
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