RESUMEN
Tissue factor is highly expressed in sub-endothelial tissue. The extracellular allosteric disulfide bond Cys186-Cys209 of human tissue factor shows high evolutionary conservation and in vitro evidence suggests that it significantly contributes to tissue factor procoagulant activity. To investigate the role of this allosteric disulfide bond in vivo, we generated a C213G mutant tissue factor mouse by replacing Cys213 of the corresponding disulfide Cys190-Cys213 in murine tissue factor. A bleeding phenotype was prominent in homozygous C213G tissue factor mice. Pre-natal lethality of 1/3rd of homozygous offspring was observed between E9.5 and E14.5 associated with placental hemorrhages. After birth, homozygous mice suffered from bleedings in different organs and reduced survival. Homozygous C213G tissue factor male mice showed higher incidence of lung bleedings and lower survival rates than females. In both sexes, C213G mutation evoked a reduced protein expression (about 10-fold) and severely reduced pro-coagulant activity (about 1000-fold). Protein glycosylation was impaired and cell membrane exposure decreased in macrophages in vivo. Single housing of homozygous C213G tissue factor males reduced the occurrence of severe bleeding and significantly improved survival, suggesting that inter-male aggressiveness might significantly account for the sex differences. These experiments show that the tissue factor allosteric disulfide bond is of crucial importance for normal in vivo expression, post-translational processing and activity of murine tissue factor. Although C213G tissue factor mice do not display the severe embryonic lethality of tissue factor knock-out mice, their postnatal bleeding phenotype emphasizes the importance of fully functional tissue factor for hemostasis.
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Disulfuros , Tromboplastina , Animales , Femenino , Hemorragia/genética , Masculino , Ratones , Mutación , Fenotipo , Embarazo , Tromboplastina/genéticaRESUMEN
The collaborative cross (CC) is a large panel of mouse-inbred lines derived from eight founder strains (NOD/ShiLtJ, NZO/HILtJ, A/J, C57BL/6J, 129S1/SvImJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ). Here, we performed a comprehensive and comparative phenotyping screening to identify phenotypic differences and similarities between the eight founder strains. In total, more than 300 parameters including allergy, behavior, cardiovascular, clinical blood chemistry, dysmorphology, bone and cartilage, energy metabolism, eye and vision, immunology, lung function, neurology, nociception, and pathology were analyzed; in most traits from sixteen females and sixteen males. We identified over 270 parameters that were significantly different between strains. This study highlights the value of the founder and CC strains for phenotype-genotype associations of many genetic traits that are highly relevant to human diseases. All data described here are publicly available from the mouse phenome database for analyses and downloads.
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Ratones Endogámicos/genética , Fenotipo , Animales , Ratones de Colaboración Cruzada/genética , Bases de Datos Genéticas , Femenino , Estudios de Asociación Genética , Genotipo , Masculino , Ratones , Sitios de Carácter Cuantitativo , Especificidad de la EspecieRESUMEN
During an ENU (N-ethyl-N-nitrosourea) mutagenesis screen, we observed a dominant small-eye mutant mouse with viable homozygotes. A corresponding mutant line was established and referred to as Aey69 (abnormality of the eye #69). Comprehensive phenotyping of the homozygous Aey69 mutants in the German Mouse Clinic revealed only a subset of statistically significant alterations between wild types and homozygous mutants. The mutation causes microphthalmia without a lens but with retinal hyperproliferation. Linkage was demonstrated to mouse chromosome 3 between the markers D3Mit188 and D3Mit11. Sequencing revealed a 358â¯A->â¯C mutation (Ile120Leu) in the Hist2h3c1 gene and a 71â¯T->â¯C (Val24Ala) mutation in the Gja8 gene. Detailed analysis of eye development in the homozygous mutant mice documented a perturbed lens development starting from the lens vesicle stage including decreasing expression of crystallins as well as of lens-specific transcription factors like PITX3 and FOXE3. In contrast, we observed an early expression of retinal progenitor cells characterized by several markers including BRN3 (retinal ganglion cells) and OTX2 (cone photoreceptors). The changes in the retina at the early embryonic stages of E11.5-E15.5 happen in parallel with apoptotic processes in the lens at the respective stages. The excessive retinal hyperproliferation is characterized by an increased level of Ki67. The hyperproliferation, however, does not disrupt the differentiation and appearance of the principal retinal cell types at postnatal stages, even if the overgrowing retina covers finally the entire bulbus of the eye. Morpholino-mediated knock-down of the hist2h3ca1 gene in zebrafish leads to a specific perturbation of lens development. When injected into zebrafish zygotes, only the mutant mouse mRNA leads to severe malformations, ranging from cyclopia to severe microphthalmia. The wild-type Hist2h3c1 mRNA can rescue the morpholino-induced defects corroborating its specific function in lens development. Based upon these data, it is concluded that the ocular function of the Hist2h3c1 gene (encoding a canonical H3.2 variant) is conserved throughout evolution. Moreover, the data highlight also the importance of Hist2h3c1 in the coordinated formation of lens and retina during eye development.
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Técnicas de Silenciamiento del Gen , Histonas/genética , Enfermedades del Cristalino/genética , Microftalmía/genética , Mutación , Animales , Cristalinas/metabolismo , Femenino , Factores de Transcripción Forkhead/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica , Hibridación in Situ , Antígeno Ki-67/metabolismo , Enfermedades del Cristalino/embriología , Enfermedades del Cristalino/metabolismo , Enfermedades del Cristalino/patología , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Microftalmía/embriología , Microftalmía/metabolismo , Microftalmía/patología , Polimorfismo de Nucleótido Simple , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción/metabolismo , Pez CebraRESUMEN
BACKGROUND: Increased levels of blood plasma urea were used as phenotypic parameter for establishing novel mouse models for kidney diseases on the genetic background of C3H inbred mice in the phenotype-driven Munich ENU mouse mutagenesis project. The phenotypically dominant mutant line HST014 was established and further analyzed. METHODS: Analysis of the causative mutation as well as the standardized, systemic phenotypic analysis of the mutant line was carried out. RESULTS: The causative mutation was detected in the potassium channel tetramerization domain containing 1 (Kctd1) gene which leads to the amino acid exchange Kctd1 I27N thereby affecting the functional BTB domain of the protein. This line is the first mouse model harboring a Kctd1 mutation. Kctd1 I27N homozygous mutant mice die perinatally. Standardized, systemic phenotypic analysis of Kctd1 I27N heterozygous mutants was carried out in the German Mouse Clinic (GMC). Systematic morphological investigation of the external physical appearance did not detect the specific alterations that are described in KCTD1 mutant human patients affected by the scalp-ear-nipple (SEN) syndrome. The main pathological phenotype of the Kctd1 I27N heterozygous mutant mice consists of kidney dysfunction and secondary effects thereof, without gross additional primary alterations in the other phenotypic parameters analyzed. Genome-wide transcriptome profiling analysis at the age of 4 months revealed about 100 differentially expressed genes (DEGs) in kidneys of Kctd1 I27N heterozygous mutants as compared to wild-type controls. CONCLUSIONS: In summary, the main alteration of the Kctd1 I27N heterozygous mutants consists in kidney dysfunction. Additional analyses in 9-21 week-old heterozygous mutants revealed only few minor effects.
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Proteínas Co-Represoras/genética , Modelos Animales de Enfermedad , Enfermedades Renales/genética , Riñón/fisiopatología , Ratones , Mutación , Animales , Femenino , Masculino , Ratones Endogámicos C3H , FenotipoRESUMEN
The human growth hormone (hGH) minigene used for transgene stabilization in mice has been recently identified to be locally expressed in the tissues where transgenes are active and associated with phenotypic alterations. Here we extend these findings by analyzing the effect of the hGH minigene in TgC6hp55 transgenic mice which express the human TNFR1 under the control of the mesenchymal cell-specific CollagenVI promoter. These mice displayed a fully penetrant phenotype characterized by growth enhancement accompanied by perturbations in metabolic, skeletal, histological and other physiological parameters. Notably, this phenotype was independent of TNF-TNFR1 signaling since the genetic ablation of either Tnf or Tradd did not rescue the phenotype. Further analyses showed that the hGH minigene was expressed in several tissues, also leading to increased hGH protein levels in the serum. Pharmacological blockade of GH signaling prevented the development of the phenotype. Our results indicate that the unplanned expression of the hGH minigene in CollagenVI expressing mesenchymal cells can lead through local and/or systemic mechanisms to enhanced somatic growth followed by a plethora of primary and/or secondary effects such as hyperphagia, hypermetabolism, disturbed glucose homeostasis, altered hematological parameters, increased bone formation and lipid accumulation in metabolically critical tissues.
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Expresión Génica , Hormona de Crecimiento Humana/genética , Fenotipo , Transgenes , Animales , Colágeno Tipo VI/genética , Femenino , Regulación de la Expresión Génica , Glucosa/metabolismo , Hormona de Crecimiento Humana/metabolismo , Humanos , Masculino , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Aging is associated with an increased risk of cardiovascular disease and death. Here we show that oral supplementation of the natural polyamine spermidine extends the lifespan of mice and exerts cardioprotective effects, reducing cardiac hypertrophy and preserving diastolic function in old mice. Spermidine feeding enhanced cardiac autophagy, mitophagy and mitochondrial respiration, and it also improved the mechano-elastical properties of cardiomyocytes in vivo, coinciding with increased titin phosphorylation and suppressed subclinical inflammation. Spermidine feeding failed to provide cardioprotection in mice that lack the autophagy-related protein Atg5 in cardiomyocytes. In Dahl salt-sensitive rats that were fed a high-salt diet, a model for hypertension-induced congestive heart failure, spermidine feeding reduced systemic blood pressure, increased titin phosphorylation and prevented cardiac hypertrophy and a decline in diastolic function, thus delaying the progression to heart failure. In humans, high levels of dietary spermidine, as assessed from food questionnaires, correlated with reduced blood pressure and a lower incidence of cardiovascular disease. Our results suggest a new and feasible strategy for protection against cardiovascular disease.
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Envejecimiento/efectos de los fármacos , Autofagia/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Corazón/efectos de los fármacos , Longevidad/efectos de los fármacos , Mitocondrias Cardíacas/efectos de los fármacos , Mitofagia/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Espermidina/farmacología , Adulto , Anciano , Envejecimiento/inmunología , Envejecimiento/metabolismo , Animales , Proteína 5 Relacionada con la Autofagia/genética , Cardiomegalia/diagnóstico por imagen , Cardiotónicos/farmacología , Enfermedades Cardiovasculares/epidemiología , Cromatografía Líquida de Alta Presión , Conectina/efectos de los fármacos , Conectina/metabolismo , Citocinas/efectos de los fármacos , Citocinas/inmunología , Diástole , Dieta/estadística & datos numéricos , Ecocardiografía , Femenino , Expresión Génica/efectos de los fármacos , Prueba de Tolerancia a la Glucosa , Corazón/diagnóstico por imagen , Insuficiencia Cardíaca , Humanos , Immunoblotting , Inflamación , Masculino , Espectrometría de Masas , Ratones , Persona de Mediana Edad , Mitocondrias Cardíacas/metabolismo , Fosforilación/efectos de los fármacos , Estudios Prospectivos , Ratas , Ratas Endogámicas Dahl , Encuestas y CuestionariosRESUMEN
The vertebrate Scube (Signal peptide, CUB, and EGF-like domain-containing protein) family consists of three independent members, Scube1-3, which encode secreted cell surface-associated membrane glycoproteins. Limited information about the general function of this gene family is available, and their roles during adulthood. Here, we present the first Scube3 mutant mouse line (Scube3N294K/N294K), which clearly shows phenotypic alterations by carrying a missense mutation in exon 8, and thus contributes to our understanding of SCUBE3 functions. We performed a detailed phenotypic characterization in the German Mouse Clinic (GMC). Scube3N294K/N294K mutants showed morphological abnormalities of the skeleton, alterations of parameters relevant for bone metabolism, changes in renal function, and hearing impairments. These findings correlate with characteristics of the rare metabolic bone disorder Paget disease of bone (PDB), associated with the chromosomal region of human SCUBE3 In addition, alterations in energy metabolism, behavior, and neurological functions were detected in Scube3N294K/N294K mice. The Scube3N294K/N294K mutant mouse line may serve as a new model for further studying the effect of impaired SCUBE3 gene function.
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Estudios de Asociación Genética , Glicoproteínas/genética , Mutación , Fenotipo , Animales , Huesos/metabolismo , Proteínas de Unión al Calcio , Mapeo Cromosómico , Modelos Animales de Enfermedad , Metabolismo Energético/genética , Exoma , Femenino , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Riñón/metabolismo , Riñón/fisiopatología , Pruebas de Función Renal , Masculino , Ratones , Ratones Noqueados , Osteítis Deformante/genética , Osteítis Deformante/metabolismo , Osteítis Deformante/patología , Esqueleto/anomalíasRESUMEN
Sphingolipids and the derived gangliosides have critical functions in spermatogenesis, thus mutations in genes involved in sphingolipid biogenesis are often associated with male infertility. We have generated a transgenic mouse line carrying an insertion in the sphingomyelin synthase gene Sms1, the enzyme which generates sphingomyelin species in the Golgi apparatus. We describe the spermatogenesis defect of Sms1-/- mice, which is characterized by sloughing of spermatocytes and spermatids, causing progressive infertility of male homozygotes. Lipid profiling revealed a reduction in several long chain unsaturated phosphatidylcholins, lysophosphatidylcholins and sphingolipids in the testes of mutants. Multi-Spectral Optoacoustic Tomography indicated blood-testis barrier dysfunction. A supplementary diet of the essential omega-3 docosahexaenoic acid and eicosapentaenoic acid diminished germ cell sloughing from the seminiferous epithelium and restored spermatogenesis and fertility in 50% of previously infertile mutants. Our findings indicate that SMS1 has a wider than anticipated role in testis polyunsaturated fatty acid homeostasis and for male fertility.
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Fertilidad , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Envejecimiento/fisiología , Empalme Alternativo , Animales , Epidídimo/efectos de los fármacos , Epidídimo/metabolismo , Ácidos Grasos Omega-3/biosíntesis , Ácidos Grasos Omega-3/farmacología , Fertilidad/efectos de los fármacos , Infertilidad Masculina/enzimología , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Ratones , Mutagénesis Insercional , Regiones Promotoras Genéticas/genética , Espermatogénesis/efectos de los fármacos , Testículo/efectos de los fármacos , Testículo/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genéticaRESUMEN
The oncoprotein Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) is overexpressed in most malignancies and is an obvious candidate target protein for future cancer therapies. However, the physiological importance of CIP2A-mediated PP2A inhibition is largely unknown. As PP2A regulates immune responses, we investigated the role of CIP2A in normal immune system development and during immune response in vivo. We show that CIP2A-deficient mice (CIP2AHOZ) present a normal immune system development and function in unchallenged conditions. However when challenged with Listeria monocytogenes, CIP2AHOZ mice display an impaired adaptive immune response that is combined with decreased frequency of both CD4+ T-cells and CD8+ effector T-cells. Importantly, the cell autonomous effect of CIP2A deficiency for T-cell activation was confirmed. Induction of CIP2A expression during T-cell activation was dependent on Zap70 activity. Thus, we reveal CIP2A as a hitherto unrecognized mediator of T-cell activation during adaptive immune response. These results also reveal CIP2AHOZ as a possible novel mouse model for studying the role of PP2A activity in immune regulation. On the other hand, the results also indicate that CIP2A targeting cancer therapies would not cause serious immunological side-effects.
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Autoantígenos/inmunología , Inmunidad/inmunología , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Activación de Linfocitos/inmunología , Proteínas de la Membrana/inmunología , Linfocitos T/inmunología , Animales , Femenino , Masculino , RatonesRESUMEN
Increased levels of blood plasma urea were used as phenotypic parameter for establishing novel mouse models for kidney diseases on the genetic background of C3H inbred mice in the phenotype-driven Munich ENU mouse mutagenesis project. The phenotypically recessive mutant line HST011 was established and further analyzed. The causative mutation was detected in the POU domain, class 3 transcription factor 3 (Pou3f3) gene, which leads to the amino acid exchange Pou3f3L423P thereby affecting the conserved homeobox domain of the protein. Pou3f3 homozygous knockout mice are published and show perinatal death. Line Pou3f3L423P is a viable mouse model harboring a homozygous Pou3f3 mutation. Standardized, systemic phenotypic analysis of homozygous mutants was carried out in the German Mouse Clinic. Main phenotypic changes were low body weight and a state of low energy stores, kidney dysfunction and secondary effects thereof including low bone mineralization, multiple behavioral and neurological defects including locomotor, vestibular, auditory and nociceptive impairments, as well as multiple subtle changes in immunological parameters. Genome-wide transcriptome profiling analysis of kidney and brain of Pou3f3L423P homozygous mutants identified significantly regulated genes as compared to wild-type controls.
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Enfermedades Renales/genética , Mutación/genética , Proteínas del Tejido Nervioso/genética , Factores del Dominio POU/genética , Animales , Modelos Animales de Enfermedad , Femenino , Genoma/genética , Homocigoto , Riñón/metabolismo , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Noqueados , Mutagénesis/genética , FenotipoRESUMEN
Tetrahydrobiopterin (BH4) is an essential cofactor for the aromatic amino acid hydroxylases, alkylglycerol monooxygenase, and nitric oxide synthases (NOS). Inborn errors of BH4 metabolism lead to severe insufficiency of brain monoamine neurotransmitters while augmentation of BH4 by supplementation or stimulation of its biosynthesis is thought to ameliorate endothelial NOS (eNOS) dysfunction, to protect from (cardio-) vascular disease and/or prevent obesity and development of the metabolic syndrome. We have previously reported that homozygous knock-out mice for the 6-pyruvolytetrahydropterin synthase (PTPS; Pts-ko/ko) mice with no BH4 biosynthesis die after birth. Here we generated a Pts-knock-in (Pts-ki) allele expressing the murine PTPS-p.Arg15Cys with low residual activity (15% of wild-type in vitro) and investigated homozygous (Pts-ki/ki) and compound heterozygous (Pts-ki/ko) mutants. All mice showed normal viability and depending on the severity of the Pts alleles exhibited up to 90% reduction of PTPS activity concomitant with neopterin elevation and mild reduction of total biopterin while blood L-phenylalanine and brain monoamine neurotransmitters were unaffected. Yet, adult mutant mice with compromised PTPS activity (i.e., Pts-ki/ko, Pts-ki/ki or Pts-ko/wt) had increased body weight and elevated intra-abdominal fat. Comprehensive phenotyping of Pts-ki/ki mice revealed alterations in energy metabolism with proportionally higher fat content but lower lean mass, and increased blood glucose and cholesterol. Transcriptome analysis indicated changes in glucose and lipid metabolism. Furthermore, differentially expressed genes associated with obesity, weight loss, hepatic steatosis, and insulin sensitivity were consistent with the observed phenotypic alterations. We conclude that reduced PTPS activity concomitant with mildly compromised BH4-biosynthesis leads to abnormal body fat distribution and abdominal obesity at least in mice. This study associates a novel single gene mutation with monogenic forms of obesity.
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Tejido Adiposo/metabolismo , Biopterinas/análogos & derivados , Distribución de la Grasa Corporal , Obesidad Abdominal/genética , Liasas de Fósforo-Oxígeno/genética , Alelos , Animales , Biopterinas/biosíntesis , Biopterinas/genética , Peso Corporal/genética , Colesterol/genética , Femenino , Genotipo , Glucosa/genética , Heterocigoto , Homocigoto , Metabolismo de los Lípidos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación/genética , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo III/genética , Fenilalanina/genética , Transcriptoma/genéticaRESUMEN
We performed exome sequencing for mutation discovery of an ENU (N-ethyl-N-nitrosourea)-derived mouse model characterized by significant elevated plasma alkaline phosphatase (ALP) activities in female and male mutant mice, originally named BAP014 (bone screen alkaline phosphatase #14). We identified a novel loss-of-function mutation within the Fam46a (family with sequence similarity 46, member A) gene (NM_001160378.1:c.469G>T, NP_001153850.1:p.Glu157*). Heterozygous mice of this mouse line (renamed Fam46a (E157*Mhda)) had significantly high ALP activities and apparently no other differences in morphology compared to wild-type mice. In contrast, homozygous Fam46a (E157*Mhda) mice showed severe morphological and skeletal abnormalities including short stature along with limb, rib, pelvis, and skull deformities with minimal trabecular bone and reduced cortical bone thickness in long bones. ALP activities of homozygous mutants were almost two-fold higher than in heterozygous mice. Fam46a is weakly expressed in most adult and embryonic tissues with a strong expression in mineralized tissues as calvaria and femur. The FAM46A protein is computationally predicted as a new member of the superfamily of nucleotidyltransferase fold proteins, but little is known about its function. Fam46a (E157*Mhda) mice are the first mouse model for a mutation within the Fam46a gene.
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Enfermedades del Desarrollo Óseo/genética , Enfermedades del Desarrollo Óseo/patología , Codón sin Sentido , Exoma , Fosfatasa Alcalina/metabolismo , Animales , Huesos/metabolismo , Huesos/patología , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Activación Enzimática , Femenino , Expresión Génica , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Masculino , Ratones , Ratones Noqueados , FenotipoRESUMEN
Fat cell metabolism has an impact on body homeostasis and its proper function. Nevertheless, the knowledge about simultaneous metabolic processes, which occur during adipogenesis and in mature adipocytes, is limited. Identification of key metabolic events associated with fat cell metabolism could be beneficial in the field of novel drug development, drug repurposing, as well as for the discovery of patterns predicting obesity risk. The main objective of our work was to provide comprehensive characterization of metabolic processes occurring during adipogenesis and in mature adipocytes. In order to globally determine crucial metabolic pathways involved in fat cell metabolism, metabolomics and transcriptomics approaches were applied. We observed significantly regulated metabolites correlating with significantly regulated genes at different stages of adipogenesis. We identified the synthesis of phosphatidylcholines, the metabolism of even and odd chain fatty acids, as well as the catabolism of branched chain amino acids (BCAA; leucine, isoleucine and valine) as key regulated pathways. Our further analysis led to identification of an enzymatic switch comprising the enzymes Hmgcs2 (3-hydroxy-3-methylglutaryl-CoA synthase) and Auh (AU RNA binding protein/enoyl-CoA hydratase) which connects leucine degradation with cholesterol synthesis. In addition, propionyl-CoA, a product of isoleucine degradation, was identified as a putative substrate for odd chain fatty acid synthesis. The uncovered crosstalks between BCAA and lipid metabolism during adipogenesis might contribute to the understanding of molecular mechanisms of obesity and have potential implications in obesity prediction.
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Adipogénesis , Perfilación de la Expresión Génica , Lípidos/biosíntesis , Metabolómica , Células 3T3-L1 , Aminoácidos de Cadena Ramificada , Animales , Colesterol/biosíntesis , Ácidos Grasos/biosíntesis , Ácidos Grasos/química , RatonesRESUMEN
We established a selection strategy to identify new models for an altered airway inflammatory response from a large compendium of mutant mouse lines that were systemically phenotyped in the German Mouse Clinic (GMC). As selection criteria we included published gene functional data, as well as immunological and transcriptome data from GMC phenotyping screens under standard conditions. Applying these criteria we identified a few from several hundred mutant mouse lines and further characterized the Cox4i2tm1Hutt, Ifit2tm1.1Ebsb, and Prdm11tm1.1ahl lines following ovalbumin (OVA) sensitization and repeated OVA airway challenge. Challenged Prdm11tm1.1ahl mice exhibited changes in B cell counts, CD4+ T cell counts, and in the number of neutrophils in bronchoalveolar lavages, whereas challenged Ifit2tm1.1Ebsb mice displayed alterations in plasma IgE, IgG1, IgG3, and IgM levels compared to the challenged wild type littermates. In contrast, challenged Cox4i2tm1Hutt mutant mice did not show alterations in the humoral or cellular immune response compared to challenged wild type mice. Transcriptome analyses from lungs of the challenged mutant mouse lines showed extensive changes in gene expression in Prdm11tm1.1ahl mice. Functional annotations of regulated genes of all three mutant mouse lines were primarily related to inflammation and airway smooth muscle (ASM) remodeling. We were thus able to define an effective selection strategy to identify new candidate genes for the predisposition to an altered airway inflammatory response under OVA challenge conditions. Similar selection strategies may be used for the analysis of additional genotype-envirotype interactions for other diseases.
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Proteínas Portadoras/genética , Mutación , Proteínas/genética , Animales , Proteínas Reguladoras de la Apoptosis , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Citocinas/sangre , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Inmunoglobulinas/sangre , Inmunoglobulinas/inmunología , Inflamación/genética , Inflamación/inmunología , Activación de Linfocitos/inmunología , Ratones , Anotación de Secuencia Molecular , Ovalbúmina/inmunología , Proteínas de Unión al ARN , Enfermedades Respiratorias/genética , Enfermedades Respiratorias/inmunología , Selección Genética , Factores de Transcripción , TranscriptomaRESUMEN
DNase I hypersensitive sites (DHSs) are a hallmark of chromatin regions containing regulatory DNA such as enhancers and promoters; however, the factors affecting the establishment and maintenance of these sites are not fully understood. We now show that HMGN1 and HMGN2, nucleosome-binding proteins that are ubiquitously expressed in vertebrate cells, maintain the DHS landscape of mouse embryonic fibroblasts (MEFs) synergistically. Loss of one of these HMGN variants led to a compensatory increase of binding of the remaining variant. Genome-wide mapping of the DHSs in Hmgn1(-/-), Hmgn2(-/-), and Hmgn1(-/-)n2(-/-) MEFs reveals that loss of both, but not a single HMGN variant, leads to significant remodeling of the DHS landscape, especially at enhancer regions marked by H3K4me1 and H3K27ac. Loss of HMGN variants affects the induced expression of stress-responsive genes in MEFs, the transcription profiles of several mouse tissues, and leads to altered phenotypes that are not seen in mice lacking only one variant. We conclude that the compensatory binding of HMGN variants to chromatin maintains the DHS landscape, and the transcription fidelity and is necessary to retain wild-type phenotypes. Our study provides insight into mechanisms that maintain regulatory sites in chromatin and into functional compensation among nucleosome binding architectural proteins.
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Sitios de Unión , Desoxirribonucleasa I/metabolismo , Elementos de Facilitación Genéticos , Proteínas HMGN/metabolismo , Animales , Línea Celular , Cromatina/metabolismo , Análisis por Conglomerados , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Proteínas HMGN/genética , Proteína HMGN1/genética , Proteína HMGN1/metabolismo , Proteína HMGN2/genética , Proteína HMGN2/metabolismo , Humanos , Ratones , Ratones Noqueados , Nucleosomas/metabolismo , Fenotipo , Regiones Promotoras Genéticas , Unión Proteica , Isoformas de Proteínas , Estrés Fisiológico/genéticaRESUMEN
Iron is essential for numerous cellular processes. For diagnostic purposes iron-related parameters in patients are assessed by clinical chemical blood analysis including the analysis of ferritin, transferrin and iron levels. Here, we retrospectively evaluated the use of these parameters in the phenotype-driven Munich N-ethyl-N-nitrosourea mouse mutagenesis project for the generation of novel animal models for human diseases. The clinical chemical blood analysis was carried out on more than 10,700 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in the plasma levels of iron-related plasma parameters. We identified animals consistently exhibiting altered plasma ferritin or transferrin values. Transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of three mutant lines with increased plasma ferritin levels. For two of these lines the causative mutations were identified in the Fth1gene and the Ireb2 gene, respectively. Thus, novel mouse models for the functional analysis of iron homeostasis were established by a phenotype-driven screen for mutant mice.
Asunto(s)
Etilnitrosourea/farmacología , Ferritinas/sangre , Mutágenos/farmacología , Animales , Secuencia de Bases , Análisis Mutacional de ADN , Femenino , Expresión Génica , Estudios de Asociación Genética , Ligamiento Genético , Pruebas Genéticas , Hierro/sangre , Masculino , Ratones Endogámicos C3H , Mutagénesis , Fenotipo , Transferrina/metabolismoRESUMEN
Previous studies have evaluated the role of miRNAs in cancer initiation and progression. MiR-34a was found to be downregulated in several tumors, including medulloblastomas. Here we employed targeted transgenesis to analyze the function of miR-34a in vivo. We generated mice with a constitutive deletion of the miR-34a gene. These mice were devoid of mir-34a expression in all analyzed tissues, but were viable and fertile. A comprehensive standardized phenotypic analysis including more than 300 single parameters revealed no apparent phenotype. Analysis of miR-34a expression in human medulloblastomas and medulloblastoma cell lines revealed significantly lower levels than in normal human cerebellum. Re-expression of miR-34a in human medulloblastoma cells reduced cell viability and proliferation, induced apoptosis and downregulated the miR-34a target genes, MYCN and SIRT1. Activation of the Shh pathway by targeting SmoA1 transgene overexpression causes medulloblastoma in mice, which is dependent on the presence and upregulation of Mycn. Analysis of miR-34a in medulloblastomas derived from ND2:SmoA1(tg) mice revealed significant suppression of miR-34a compared to normal cerebellum. Tumor incidence was significantly increased and tumor formation was significantly accelerated in mice transgenic for SmoA1 and lacking miR-34a. Interestingly, Mycn and Sirt1 were strongly expressed in medulloblastomas derived from these mice. We here demonstrate that miR-34a is dispensable for normal development, but that its loss accelerates medulloblastomagenesis. Strategies aiming to re-express miR-34a in tumors could, therefore, represent an efficient therapeutic option.
Asunto(s)
Neoplasias Cerebelosas/patología , Cerebelo/metabolismo , Meduloblastoma/patología , MicroARNs/genética , MicroARNs/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/metabolismo , Regulación Neoplásica de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Meduloblastoma/genética , Meduloblastoma/metabolismo , Ratones , Ratones Transgénicos , Fenotipo , Transducción de SeñalRESUMEN
The paracaspase Malt1 is a central regulator of antigen receptor signaling that is frequently mutated in human lymphoma. As a scaffold, it assembles protein complexes for NF-κB activation, and its proteolytic domain cleaves negative NF-κB regulators for signal enforcement. Still, the physiological functions of Malt1-protease are unknown. We demonstrate that targeted Malt1-paracaspase inactivation induces a lethal inflammatory syndrome with lymphocyte-dependent neurodegeneration in vivo. Paracaspase activity is essential for regulatory T cell (Treg) and innate-like B cell development, but it is largely dispensable for overcoming Malt1-dependent thresholds for lymphocyte activation. In addition to NF-κB inhibitors, Malt1 cleaves an entire set of mRNA stability regulators, including Roquin-1, Roquin-2, and Regnase-1, and paracaspase inactivation results in excessive interferon gamma (IFNγ) production by effector lymphocytes that drive pathology. Together, our results reveal distinct threshold and modulatory functions of Malt1 that differentially control lymphocyte differentiation and activation pathways and demonstrate that selective paracaspase blockage skews systemic immunity toward destructive autoinflammation.
Asunto(s)
Autoinmunidad , Caspasas/metabolismo , Inflamación/inmunología , Inflamación/patología , Proteínas de Neoplasias/metabolismo , Animales , Linfocitos B/inmunología , Caspasas/deficiencia , Diferenciación Celular/inmunología , Regulación de la Expresión Génica , Homeostasis/inmunología , Humanos , Inmunidad Mucosa/inmunología , Interferón gamma/biosíntesis , Activación de Linfocitos/inmunología , Ratones Mutantes , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Proteínas de Neoplasias/deficiencia , Degeneración Nerviosa/inmunología , Degeneración Nerviosa/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunologíaRESUMEN
Uromodulin-associated kidney disease (UAKD) is a hereditary progressive renal disease which can lead to renal failure and requires renal replacement therapy. UAKD belongs to the endoplasmic reticulum storage diseases due to maturation defect of mutant uromodulin and its retention in the enlarged endoplasmic reticulum in the cells of the thick ascending limb of Henle's loop (TALH). Dysfunction of TALH represents the key pathogenic mechanism of UAKD causing the clinical symptoms of this disease. However, the molecular alterations underlying UAKD are not well understood. In this study, transcriptome profiling of whole kidneys of two mouse models of UAKD, UmodA227T and UmodC93F, was performed. Genes differentially abundant in UAKD affected kidneys of both Umod mutant lines at different disease stages were identified and verified by RT-qPCR. Additionally, differential protein abundances of SCD1 and ANGPTL7 were validated by immunohistochemistry and Western blot analysis. ANGPTL7 expression was down-regulated in TALH cells of Umod mutant mice which is the site of the mutant uromodulin maturation defect. SCD1 was expressed selectively in the S3 segment of proximal tubule cells, and SCD1 abundance was increased in UAKD affected kidneys. This finding demonstrates that a cross talk between two functionally distinct tubular segments of the kidney, the TALH segment and the S3 segment of proximal tubule, exists.
Asunto(s)
Gota/metabolismo , Hiperuricemia/metabolismo , Enfermedades Renales/metabolismo , Túbulos Renales Proximales/metabolismo , Asa de la Nefrona/metabolismo , Estearoil-CoA Desaturasa/genética , Uromodulina/genética , Proteína 7 Similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina , Angiopoyetinas/genética , Angiopoyetinas/metabolismo , Animales , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Gota/genética , Gota/patología , Hiperuricemia/genética , Hiperuricemia/patología , Enfermedades Renales/genética , Enfermedades Renales/patología , Túbulos Renales Proximales/patología , Asa de la Nefrona/patología , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Estearoil-CoA Desaturasa/metabolismo , Uromodulina/metabolismoRESUMEN
Mutations in Peroxidasin (PXDN) cause severe inherited eye disorders in humans, such as congenital cataract, corneal opacity and developmental glaucoma. The role of peroxidasin during eye development is poorly understood. Here, we describe the first Pxdn mouse mutant which was induced by ENU (N-ethyl-N-nitrosourea) and led to a recessive phenotype. Sequence analysis of cDNA revealed a T3816A mutation resulting in a premature stop codon (Cys1272X) in the peroxidase domain. This mutation causes severe anterior segment dysgenesis and microphthalmia resembling the manifestations in patients with PXDN mutations. The proliferation and differentiation of the lens is disrupted in association with aberrant expression of transcription factor genes (Pax6 and Foxe3) in mutant eyes. Additionally, Pxdn is involved in the consolidation of the basement membrane and lens epithelium adhesion in the ocular lens. Lens material including γ-crystallin is extruded into the anterior and posterior chamber due to local loss of structural integrity of the lens capsule as a secondary damage to the anterior segment development leading to congenital ocular inflammation. Moreover, Pxdn mutants exhibited an early-onset glaucoma and progressive retinal dysgenesis. Transcriptome profiling revealed that peroxidasin affects the transcription of developmental and eye disease-related genes at early eye development. These findings suggest that peroxidasin is necessary for cell proliferation and differentiation and for basement membrane consolidation during eye development. Our studies provide pathogenic mechanisms of PXDN mutation-induced congenital eye diseases.