RESUMEN
WW domain-containing oxidoreductase (WWOX) is highly conserved in both human and murine. WWOX spans the second most common human chromosomal fragile site, FRA16D, and is commonly inactivated in multiple human cancers. Modeling WWOX inactivation in mice revealed a complex phenotype including postnatal lethality, defects in bone metabolism and steroidogenesis and tumor suppressor function resulting in osteosarcomas. For better understanding of WWOX roles in different tissues at distinct stages of development and in pathological conditions, Wwox conditional knockout mice were generated in which loxp sites flank exon 1 in the Wwox allele. We demonstrated that Cre-mediated recombination using EIIA-Cre, a Cre line expressed in germline, results in postnatal lethality by age of 3 weeks and decreased bone mineralization resembling total ablation of WWOX as in conventional null mice. This animal model will be useful to study distinct roles of WWOX in multiple tissues at different ages.
Asunto(s)
Genes Supresores de Tumor , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/genética , Animales , Calcificación Fisiológica/genética , Calcificación Fisiológica/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica , Marcación de Gen , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Osteosarcoma/genética , Osteosarcoma/patología , Oxidorreductasas/fisiología , Fenotipo , Esteroides/biosíntesis , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/fisiología , Oxidorreductasa que Contiene Dominios WWRESUMEN
The prion protein (PrP) is essential for the pathogenesis of prion disease. PrP has been detected in the cytosol of neurons and transgenic mice expressing PrP in the cytosol (cyPrP) under a pan-neuronal promoter developed rapid cerebellar granule neuron degeneration. Yet, it remains unclear whether cyPrP is capable to cause toxicity in other neuronal populations. Here, we report that transgenic mice expressing cyPrP in the forebrain neurons developed behavioral abnormalities including clasping and hyperactivity. These mice had reduced thickness in cortex and developed astrogliosis in hippocampal and cortical regions. Moreover, cyPrP in these mice was recognized by the A11 anti-oligomer antibody and was associated with the hydrophobic lipid core of membranes, indicating that cyPrP oligomer caused membrane perturbation contributes to cyPrP neurotoxicity. Together, our results clearly revealed that cyPrP is able to cause toxicity in different neuronal populations, supporting a role of cyPrP in PrP-mediated neurodegenerative disorders.
Asunto(s)
Síndromes de Neurotoxicidad/metabolismo , Proteínas PrPC/patogenicidad , Enfermedades por Prión/patología , Prosencéfalo/patología , Animales , Conducta Animal , Corteza Cerebral/patología , Citoplasma/metabolismo , Hipocampo/patología , Homocigoto , Membrana Dobles de Lípidos/metabolismo , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Síndromes de Neurotoxicidad/patología , Proteínas PrPC/metabolismoRESUMEN
Evidence of dysregulation of the CREB/CRE transcriptional pathway in animal models of Huntington's disease (HD) suggests that strategies designed to augment CRE-mediated transcription may be of therapeutic value. Here, we investigated the consequences of CREB activation and repression in chemical and transgenic mouse models of HD. In the 3-nitropropionic acid (3-NP) model, CREB phospho-activation in the striatum was potently repressed within the neurotoxic "core" region prior to cell death. Conversely, marked expression of phospho-CREB, as well the CREB-regulated cytoprotective gene Bcl-2, was detected in the "penumbral" region. To examine potential contributory roles for the CREB/CRE transcriptional pathway in striatal degeneration, we used both CREB loss- (A-CREB) and gain- (VP16-CREB) of-function transgenic mouse strains. 3-NP-induced striatal lesion size and motor dysfunction were significantly increased in A-CREB mice compared to controls. Conversely, striatal damage and motor deficits were diminished in VP16-CREB mice. Furthermore, transgenic A-CREB significantly accelerated motor impairment in the YAC128 mouse model of HD. Together, these results indicate that CREB functionality is lost during the early stages of striatal cell stress and that the repression of CREB-mediated transcription contributes to the pathogenic process.
Asunto(s)
Cuerpo Estriado/patología , Cuerpo Estriado/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Modelos Animales de Enfermedad , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Animales , Células Cultivadas , Cuerpo Estriado/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/antagonistas & inhibidores , Enfermedad de Huntington/genética , Enfermedad de Huntington/fisiopatología , Masculino , Ratones , Ratones Transgénicos , Nitrocompuestos/toxicidad , Propionatos/toxicidad , Ratas , Ratas Sprague-Dawley , Transcripción Genética/efectos de los fármacos , Transcripción Genética/fisiologíaRESUMEN
Pronuclear microinjection remains the most widely used method for the germline modification of mice and other species. The method is conceptually quite simple and at least in rodents can produce transgenic offspring with relatively high efficiency. Here, we describe the various components of the production of transgenic mice including a detailed list of materials and equipment. We outline in detail the preparation of animals, the retrieval, culture and transfer of embryos, the preparation of DNA, and the microinjection process. We have added a substantial collection of notes with helpful suggestions that reflect the many years of experience we have using this technology and our continuing efforts to improve animal welfare and the efficiency of producing transgenics.
Asunto(s)
Núcleo Celular/genética , ADN/administración & dosificación , Ratones Transgénicos/crecimiento & desarrollo , Microinyecciones/métodos , Animales , Técnicas de Cultivo de Embriones , Transferencia de Embrión , Femenino , Técnicas de Transferencia de Gen , Masculino , RatonesRESUMEN
Interleukin-1 (IL-1) has been implicated as a critical mediator of neuroimmune communication. In the brain, the functional receptor for IL-1, type 1 IL-1 receptor (IL-1R1), is localized primarily to the endothelial cells. In this study, we created an endothelial-specific IL-1R1 knockdown model to test the role of endothelial IL-1R1 in mediating the effects of IL-1. Neuronal activation in the hypothalamus was measured by c-fos expression in the paraventricular nucleus and the ventromedial preoptic area. In addition, two specific sickness symptoms, febrile response and reduction of locomotor activity, were studied. Intracerebroventricular injection of IL-1 induced leukocyte infiltration into the CNS, activation of hypothalamic neurons, fever, and reduced locomotor activity in normal mice. Endothelial-specific knockdown of IL-1R1 abrogated all these responses. Intraperitoneal injection of IL-1 also induced neuronal activation in the hypothalamus, fever, and reduced locomotor activity, without inducing leukocyte infiltration into the brain. Endothelial-specific knockdown of IL-1R1 suppressed intraperitoneal IL-1-induced fever, but not the induction of c-fos in hypothalamus. When IL-1 was given intravenously, endothelial knockdown of IL-1R1 abolished intravenous IL-1-induced CNS activation and the two monitored sickness symptoms. In addition, endothelial-specific knockdown of IL-1R1 blocked the induction of cyclooxygenase-2 expression induced by all three routes of IL-1 administration. These results show that the effects of intravenous and intracerebroventricular IL-1 are mediated by endothelial IL-1R1, whereas the effects of intraperitoneal IL-1 are partially dependent on endothelial IL-1R1.
Asunto(s)
Sistema Nervioso Central/efectos de los fármacos , Células Endoteliales/metabolismo , Factores Inmunológicos/administración & dosificación , Interleucina-1/administración & dosificación , Actividad Motora/fisiología , Receptores Tipo I de Interleucina-1/fisiología , Animales , Ciclooxigenasa 2/biosíntesis , Fiebre/fisiopatología , Genes fos/fisiología , Hipotálamo/efectos de los fármacos , Ratones , Ratones Transgénicos , Actividad Motora/efectos de los fármacos , Neuroinmunomodulación/efectos de los fármacos , Receptores Tipo I de Interleucina-1/efectos de los fármacosRESUMEN
Pcp2(L7) is a Purkinje cell-specific GoLoco domain protein that modulates activation of Galphai/o proteins by G protein-coupled receptors. A likely downstream effector of this pathway is the P-type Ca(2+) channel, and thereby, the intrinsic electrophysiology of Purkinje cells could be modulated by Pcp2(L7). It has long been known that the Pcp2(L7) mRNA is abundantly localized in dendrites, suggesting the possibility of distal synthesis and local changes in levels of the protein. As a first step to uncover the trafficking and translational mechanisms for this mRNA, we have begun identifying the cis-acting sequences important for its localization in dendrites. Using expression of modified transgenes in vivo, we show that the 3'UTR, only 65 bases long, is necessary in this process.
Asunto(s)
Regiones no Traducidas 3'/fisiología , Dendritas/fisiología , Células de Purkinje/fisiología , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/fisiología , Regiones no Traducidas 3'/genética , Animales , Cartilla de ADN , Amplificación de Genes , Hibridación in Situ , Ratones , Ratones Transgénicos , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Viral/genética , Virus 40 de los Simios/genéticaRESUMEN
AIMS: Prior work suggests that ischemic preconditioning increases the level of CD39 in the heart and contributes to cardiac protection. Therefore, we examined if targeted cardiac expression of CD39 protects against myocardial injury. MAIN METHODS: Mice with cardiac-specific expression of human CD39 (αMHC/hCD39-Tg) were generated, characterized and subjected to left coronary artery ischemia-reperfusion injury and infarct size at 24h following injury quantified. KEY FINDINGS: αMHC/hCD39-Tg mice have increased in cardiac ATPase and ADPase activity compared to WT littermates. The increased activity in αMHC/hCD39-mice was inhibited by the CD39 antagonist sodium polyoxotungstate (POM-1). Measurement of basal cardiac function by echocardiography revealed that αMHC/hCD39-Tg mice have a lower resting heart rate and increased stroke volume. In response to myocardial ischemia, systolic and diastolic function was better preserved in αMHC/hCD39-Tg compared to WT mice. Comparison of Tau also revealed preserved cardiac relaxation during ischemia in αMHC/hCD39-Tg hearts. Assessment of myocardial infarct size in response to 60min of ischemia and 24h of reperfusion demonstrated a significant reduction in infarct size in αMHC/hCD39-Tg hearts. Analysis of isolated cardiomyocytes revealed no basal difference in calcium transients between WT and αMHC/hCD39-Tg cardiomyocytes. However, in response to isoproterenol stimulation, there was a trend toward lower calcium transients in αMHC/hCD39 cardiomyocytes suggesting less calcium accumulation in response to metabolic stress. SIGNIFICANCE: Cardiac-specific expression of CD39 reduces myocardial dysfunction and infarct size following ischemia-reperfusion injury. Increasing nucleotidase expression in the heart may be a novel approach to protect the heart from ischemic injury.
Asunto(s)
Antígenos CD/genética , Apirasa/genética , Expresión Génica , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/complicaciones , Animales , Calcio/metabolismo , Modelos Animales de Enfermedad , Ecocardiografía , Frecuencia Cardíaca/genética , Humanos , Isoproterenol/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Infarto del Miocardio/genética , Daño por Reperfusión Miocárdica/genética , Miocitos Cardíacos/metabolismo , Estrés Fisiológico/genética , Volumen Sistólico/genéticaRESUMEN
CD24 is a cell surface glycoprotein that is expressed on both immune cells and cells of the CNS. We have previously shown that CD24 is required for the induction of experimental autoimmune encephalomyelitis (EAE), an experimental model for the human disease multiple sclerosis (MS). The development of EAE requires CD24 expression on both T cells and non-T host cells in the CNS. To understand the role of CD24 on the resident cells in the CNS during EAE development, we created CD24 bone marrow chimeras and transgenic mice in which CD24 expression was under the control of a glial fibrillary acidic protein promotor (AstroCD24TG mice). We showed that mice lacking CD24 expression on the CNS resident cells developed a mild form of EAE; in contrast, mice with overexpression of CD24 in the CNS developed severe EAE. Compared with nontransgenic mice, the CNS of AstroCD24TG mice had higher expression of cytokine genes such as IL-17 and demyelination-associated marker P8; the CNS of AstroCD24TG mice accumulated higher numbers of Th17 and total CD4+ T cells, whereas CD4+ T cells underwent more proliferation during EAE development. Expression of CD24 in CD24-deficient astrocytes also enhanced their costimulatory activity to myelin oligodendrocyte glycoprotein-specific, TCR-transgenic 2D2 T cells. Thus, CD24 on the resident cells in the CNS enhances EAE development via costimulation of encephalitogenic T cells. Because CD24 is increased drastically on resident cells in the CNS during EAE, our data have important implications for CD24-targeted therapy of MS.
Asunto(s)
Adyuvantes Inmunológicos/fisiología , Antígeno CD24/fisiología , Sistema Nervioso Central/citología , Sistema Nervioso Central/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Adyuvantes Inmunológicos/genética , Adyuvantes Inmunológicos/metabolismo , Secuencia de Aminoácidos , Animales , Antígeno CD24/genética , Antígeno CD24/metabolismo , Proliferación Celular , Células Cultivadas , Sistema Nervioso Central/metabolismo , Citocinas/biosíntesis , Citocinas/genética , Progresión de la Enfermedad , Encefalomielitis Autoinmune Experimental/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Esclerosis Múltiple/terapiaRESUMEN
Cancer stem cells (CSCs) have been identified in hematopoietic and solid tumors. However, their precursors-namely, precancerous stem cells (pCSCs) -have not been characterized. Here we experimentally define the pCSCs that have the potential for both benign and malignant differentiation, depending on environmental cues. While clonal pCSCs can develop into various types of tissue cells in immunocompetent mice without developing into cancer, they often develop, however, into leukemic or solid cancers composed of various types of cancer cells in immunodeficient mice. The progress of the pCSCs to cancers is associated with the up-regulation of c-kit and Sca-1, as well as with lineage markers. Mechanistically, the pCSCs are regulated by the PIWI/AGO family gene called piwil2. Our results provide clear evidence that a single clone of pCSCs has the potential for both benign and malignant differentiation, depending on the environmental cues. We anticipate pCSCs to be a novel target for the early detection, prevention, and therapy of cancers.