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1.
Nat Immunol ; 21(1): 86-100, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31844327

RESUMO

By developing a high-density murine immunophenotyping platform compatible with high-throughput genetic screening, we have established profound contributions of genetics and structure to immune variation (http://www.immunophenotype.org). Specifically, high-throughput phenotyping of 530 unique mouse gene knockouts identified 140 monogenic 'hits', of which most had no previous immunologic association. Furthermore, hits were collectively enriched in genes for which humans show poor tolerance to loss of function. The immunophenotyping platform also exposed dense correlation networks linking immune parameters with each other and with specific physiologic traits. Such linkages limit freedom of movement for individual immune parameters, thereby imposing genetically regulated 'immunologic structures', the integrity of which was associated with immunocompetence. Hence, we provide an expanded genetic resource and structural perspective for understanding and monitoring immune variation in health and disease.


Assuntos
Infecções por Enterobacteriaceae/imunologia , Variação Genética/genética , Ensaios de Triagem em Larga Escala/métodos , Imunofenotipagem/métodos , Infecções por Salmonella/imunologia , Animais , Citrobacter/imunologia , Infecções por Enterobacteriaceae/microbiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Salmonella/imunologia , Infecções por Salmonella/microbiologia
2.
Cell ; 154(2): 452-64, 2013 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-23870131

RESUMO

Mutations in whole organisms are powerful ways of interrogating gene function in a realistic context. We describe a program, the Sanger Institute Mouse Genetics Project, that provides a step toward the aim of knocking out all genes and screening each line for a broad range of traits. We found that hitherto unpublished genes were as likely to reveal phenotypes as known genes, suggesting that novel genes represent a rich resource for investigating the molecular basis of disease. We found many unexpected phenotypes detected only because we screened for them, emphasizing the value of screening all mutants for a wide range of traits. Haploinsufficiency and pleiotropy were both surprisingly common. Forty-two percent of genes were essential for viability, and these were less likely to have a paralog and more likely to contribute to a protein complex than other genes. Phenotypic data and more than 900 mutants are openly available for further analysis. PAPERCLIP:


Assuntos
Técnicas Genéticas , Camundongos Knockout , Fenótipo , Animais , Doença/genética , Modelos Animais de Doenças , Feminino , Genes Essenciais , Estudo de Associação Genômica Ampla , Masculino , Camundongos
3.
PLoS Genet ; 20(4): e1011228, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38598567

RESUMO

The laboratory mouse has served as the premier animal model system for both basic and preclinical investigations for over a century. However, laboratory mice capture only a subset of the genetic variation found in wild mouse populations, ultimately limiting the potential of classical inbred strains to uncover phenotype-associated variants and pathways. Wild mouse populations are reservoirs of genetic diversity that could facilitate the discovery of new functional and disease-associated alleles, but the scarcity of commercially available, well-characterized wild mouse strains limits their broader adoption in biomedical research. To overcome this barrier, we have recently developed, sequenced, and phenotyped a set of 11 inbred strains derived from wild-caught Mus musculus domesticus. Each of these "Nachman strains" immortalizes a unique wild haplotype sampled from one of five environmentally distinct locations across North and South America. Whole genome sequence analysis reveals that each strain carries between 4.73-6.54 million single nucleotide differences relative to the GRCm39 mouse reference, with 42.5% of variants in the Nachman strain genomes absent from current classical inbred mouse strain panels. We phenotyped the Nachman strains on a customized pipeline to assess the scope of disease-relevant neurobehavioral, biochemical, physiological, metabolic, and morphological trait variation. The Nachman strains exhibit significant inter-strain variation in >90% of 1119 surveyed traits and expand the range of phenotypic diversity captured in classical inbred strain panels. These novel wild-derived inbred mouse strain resources are set to empower new discoveries in both basic and preclinical research.


Assuntos
Variação Genética , Camundongos Endogâmicos , Fenótipo , Animais , Camundongos , Camundongos Endogâmicos/genética , Genômica/métodos , Animais Selvagens/genética , Genoma/genética , Polimorfismo de Nucleotídeo Único , Haplótipos , Sequenciamento Completo do Genoma
4.
Nature ; 555(7697): 463-468, 2018 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-29539633

RESUMO

Large-scale phenotyping efforts have demonstrated that approximately 25-30% of mouse gene knockouts cause intrauterine lethality. Analysis of these mutants has largely focused on the embryo and not the placenta, despite the crucial role of this extraembryonic organ for developmental progression. Here we screened 103 embryonic lethal and sub-viable mouse knockout lines from the Deciphering the Mechanisms of Developmental Disorders program for placental phenotypes. We found that 68% of knockout lines that are lethal at or after mid-gestation exhibited placental dysmorphologies. Early lethality (embryonic days 9.5-14.5) is almost always associated with severe placental malformations. Placental defects correlate strongly with abnormal brain, heart and vascular development. Analysis of mutant trophoblast stem cells and conditional knockouts suggests that a considerable number of factors that cause embryonic lethality when ablated have primary gene function in trophoblast cells. Our data highlight the hugely under-appreciated importance of placental defects in contributing to abnormal embryo development and suggest key molecular nodes that govern placenta formation.


Assuntos
Perda do Embrião/genética , Perda do Embrião/patologia , Mutação , Placenta/patologia , Placentação/genética , Animais , Feminino , Camundongos , Camundongos Knockout , Gravidez , Células-Tronco/metabolismo , Células-Tronco/patologia , Trofoblastos/metabolismo , Trofoblastos/patologia
5.
Mamm Genome ; 34(2): 180-199, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37294348

RESUMO

Reference ranges provide a powerful tool for diagnostic decision-making in clinical medicine and are enormously valuable for understanding normality in pre-clinical scientific research that uses in vivo models. As yet, there are no published reference ranges for electrocardiography (ECG) in the laboratory mouse. The first mouse-specific reference ranges for the assessment of electrical conduction are reported herein generated from an ECG dataset of unprecedented scale. International Mouse Phenotyping Consortium data from over 26,000 conscious or anesthetized C57BL/6N wildtype control mice were stratified by sex and age to develop robust ECG reference ranges. Interesting findings include that heart rate and key elements from the ECG waveform (RR-, PR-, ST-, QT-interval, QT corrected, and QRS complex) demonstrate minimal sexual dimorphism. As expected, anesthesia induces a decrease in heart rate and was shown for both inhalation (isoflurane) and injectable (tribromoethanol) anesthesia. In the absence of pharmacological, environmental, or genetic challenges, we did not observe major age-related ECG changes in C57BL/6N-inbred mice as the differences in the reference ranges of 12-week-old compared to 62-week-old mice were negligible. The generalizability of the C57BL/6N substrain reference ranges was demonstrated by comparison with ECG data from a wide range of non-IMPC studies. The close overlap in data from a wide range of mouse strains suggests that the C57BL/6N-based reference ranges can be used as a robust and comprehensive indicator of normality. We report a unique ECG reference resource of fundamental importance for any experimental study of cardiac function in mice.


Assuntos
Eletrocardiografia , Técnicas Eletrofisiológicas Cardíacas , Camundongos , Animais , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos
7.
BMC Biol ; 20(1): 67, 2022 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-35296311

RESUMO

BACKGROUND: Mice carrying targeted mutations are important for investigating gene function and the role of genes in disease, but off-target mutagenic effects associated with the processes of generating targeted alleles, for instance using Crispr, and culturing embryonic stem cells, offer opportunities for spontaneous mutations to arise. Identifying spontaneous mutations relies on the detection of phenotypes segregating independently of targeted alleles, and having a broad estimate of the level of mutations generated by intensive breeding programmes is difficult given that many phenotypes are easy to miss if not specifically looked for. Here we present data from a large, targeted knockout programme in which mice were analysed through a phenotyping pipeline. Such spontaneous mutations segregating within mutant lines may confound phenotypic analyses, highlighting the importance of record-keeping and maintaining correct pedigrees. RESULTS: Twenty-five lines out of 1311 displayed different deafness phenotypes that did not segregate with the targeted allele. We observed a variety of phenotypes by Auditory Brainstem Response (ABR) and behavioural assessment and isolated eight lines showing early-onset severe progressive hearing loss, later-onset progressive hearing loss, low frequency hearing loss, or complete deafness, with vestibular dysfunction. The causative mutations identified include deletions, insertions, and point mutations, some of which involve new genes not previously associated with deafness while others are new alleles of genes known to underlie hearing loss. Two of the latter show a phenotype much reduced in severity compared to other mutant alleles of the same gene. We investigated the ES cells from which these lines were derived and determined that only one of the 8 mutations could have arisen in the ES cell, and in that case, only after targeting. Instead, most of the non-segregating mutations appear to have occurred during breeding of mutant mice. In one case, the mutation arose within the wildtype colony used for expanding mutant lines. CONCLUSIONS: Our data show that spontaneous mutations with observable effects on phenotype are a common side effect of intensive breeding programmes, including those underlying targeted mutation programmes. Such spontaneous mutations segregating within mutant lines may confound phenotypic analyses, highlighting the importance of record-keeping and maintaining correct pedigrees.


Assuntos
Surdez , Perda Auditiva , Alelos , Animais , Surdez/genética , Perda Auditiva/genética , Camundongos , Mutagênese , Mutação
8.
Genome Res ; 29(3): 494-505, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30659012

RESUMO

Transgenesis has been a mainstay of mouse genetics for over 30 yr, providing numerous models of human disease and critical genetic tools in widespread use today. Generated through the random integration of DNA fragments into the host genome, transgenesis can lead to insertional mutagenesis if a coding gene or an essential element is disrupted, and there is evidence that larger scale structural variation can accompany the integration. The insertion sites of only a tiny fraction of the thousands of transgenic lines in existence have been discovered and reported, due in part to limitations in the discovery tools. Targeted locus amplification (TLA) provides a robust and efficient means to identify both the insertion site and content of transgenes through deep sequencing of genomic loci linked to specific known transgene cassettes. Here, we report the first large-scale analysis of transgene insertion sites from 40 highly used transgenic mouse lines. We show that the transgenes disrupt the coding sequence of endogenous genes in half of the lines, frequently involving large deletions and/or structural variations at the insertion site. Furthermore, we identify a number of unexpected sequences in some of the transgenes, including undocumented cassettes and contaminating DNA fragments. We demonstrate that these transgene insertions can have phenotypic consequences, which could confound certain experiments, emphasizing the need for careful attention to control strategies. Together, these data show that transgenic alleles display a high rate of potentially confounding genetic events and highlight the need for careful characterization of each line to assure interpretable and reproducible experiments.


Assuntos
Variação Estrutural do Genoma , Recombinação Genética , Transgenes , Animais , Células Cultivadas , Técnicas de Genotipagem/métodos , Camundongos , Camundongos Transgênicos , Mutagênese Insercional , Técnicas de Amplificação de Ácido Nucleico/métodos , Fenótipo
9.
J Anat ; 240(1): 11-22, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34435363

RESUMO

Approximately one-third of randomly produced knockout mouse lines produce homozygous offspring, which fail to survive the perinatal period. The majority of these die around or after embryonic day (E)14.5, presumably from cardiovascular insufficiency. For diagnosing structural abnormalities underlying death and diseases and for researching gene function, the phenotype of these individuals has to be analysed. This makes the creation of reference data, which define normal anatomy and normal variations the highest priority. While such data do exist for the heart and arteries, they are still missing for the venous system. Here we provide high-quality descriptive and metric information on the normal anatomy of the venous system of E14.5 embryos. Using high-resolution digital volume data and 3D models from 206 genetically normal embryos, bred on the C57BL/6N background, we present precise descriptive and metric information of the venous system as it presents itself in each of the six developmental stages of E14.5. The resulting data shed new light on the maturation and remodelling of the venous system at transition of embryo to foetal life and provide a reference that can be used for detecting venous abnormalities in mutants. To explore this capacity, we analysed the venous phenotype of embryos from 7 knockout lines (Atp11a, Morc2a, 1700067K01Rik, B9d2, Oaz1, Celf4 and Coro1c). Careful comparisons enabled the diagnosis of not only simple malformations, such as dual inferior vena cava, but also complex and subtle abnormalities, which would have escaped diagnosis in the absence of detailed, stage-specific referenced data.


Assuntos
Embrião de Mamíferos , Animais , Feminino , Deleção de Genes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Gravidez
10.
PLoS Biol ; 17(4): e3000194, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30973865

RESUMO

Adult-onset hearing loss is very common, but we know little about the underlying molecular pathogenesis impeding the development of therapies. We took a genetic approach to identify new molecules involved in hearing loss by screening a large cohort of newly generated mouse mutants using a sensitive electrophysiological test, the auditory brainstem response (ABR). We review here the findings from this screen. Thirty-eight unexpected genes associated with raised thresholds were detected from our unbiased sample of 1,211 genes tested, suggesting extreme genetic heterogeneity. A wide range of auditory pathophysiologies was found, and some mutant lines showed normal development followed by deterioration of responses, revealing new molecular pathways involved in progressive hearing loss. Several of the genes were associated with the range of hearing thresholds in the human population and one, SPNS2, was involved in childhood deafness. The new pathways required for maintenance of hearing discovered by this screen present new therapeutic opportunities.


Assuntos
Percepção Auditiva/genética , Potenciais Evocados Auditivos do Tronco Encefálico/genética , Perda Auditiva/genética , Estimulação Acústica/métodos , Adulto , Animais , Proteínas de Transporte de Ânions/genética , Criança , Fenômenos Eletrofisiológicos/genética , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Feminino , Estudos de Associação Genética , Audição/genética , Perda Auditiva/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL
11.
J Immunol ; 205(8): 2026-2038, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32938729

RESUMO

It has become increasingly appreciated that autoimmune responses against neuronal components play an important role in type 1 diabetes (T1D) pathogenesis. In fact, a large proportion of islet-infiltrating B lymphocytes in the NOD mouse model of T1D produce Abs directed against the neuronal type III intermediate filament protein peripherin. NOD-PerIg mice are a previously developed BCR-transgenic model in which virtually all B lymphocytes express the H and L chain Ig molecules from the intra-islet-derived anti-peripherin-reactive hybridoma H280. NOD-PerIg mice have accelerated T1D development, and PerIg B lymphocytes actively proliferate within islets and expand cognitively interactive pathogenic T cells from a pool of naive precursors. We now report adoptively transferred T cells or whole splenocytes from NOD-PerIg mice expectedly induce T1D in NOD.scid recipients but, depending on the kinetics of disease development, can also elicit a peripheral neuritis (with secondary myositis). This neuritis was predominantly composed of CD4+ and CD8+ T cells. Ab depletion studies showed neuritis still developed in the absence of NOD-PerIg CD8+ T cells but required CD4+ T cells. Surprisingly, sciatic nerve-infiltrating CD4+ cells had an expansion of IFN-γ- and TNF-α- double-negative cells compared with those within both islets and spleen. Nerve and islet-infiltrating CD4+ T cells also differed by expression patterns of CD95, PD-1, and Tim-3. Further studies found transitory early B lymphocyte depletion delayed T1D onset in a portion of NOD-PerIg mice, allowing them to survive long enough to develop neuritis outside of the transfer setting. Together, this study presents a new model of peripherin-reactive B lymphocyte-dependent autoimmune neuritis.


Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Tecido Nervoso , Neurite Autoimune Experimental , Pâncreas , Animais , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Tecido Nervoso/imunologia , Tecido Nervoso/patologia , Neurite Autoimune Experimental/genética , Neurite Autoimune Experimental/imunologia , Neurite Autoimune Experimental/patologia , Pâncreas/imunologia , Pâncreas/patologia
12.
Nature ; 537(7621): 508-514, 2016 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-27626380

RESUMO

Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.


Assuntos
Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Genes Essenciais/genética , Genes Letais/genética , Mutação/genética , Fenótipo , Animais , Sequência Conservada/genética , Doença , Estudo de Associação Genômica Ampla , Ensaios de Triagem em Larga Escala , Humanos , Imageamento Tridimensional , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Penetrância , Polimorfismo de Nucleotídeo Único/genética , Homologia de Sequência
13.
Mol Pain ; 16: 1744806920958596, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32955381

RESUMO

The discovery and development of new and potentially nonaddictive pain therapeutics requires rapid, yet clinically relevant assays of nociception in preclinical models. A reliable and scalable automated scoring system for nocifensive behavior of mice in the formalin assay would dramatically lower the time and labor costs associated with experiments and reduce experimental variability. Here, we present a method that exploits machine learning techniques for video recordings that consists of three components: key point detection, per frame feature extraction using these key points, and classification of behavior using the GentleBoost algorithm. This approach to automation is flexible as different model classifiers or key points can be used with only small losses in accuracy. The adopted system identified the behavior of licking/biting of the hind paw with an accuracy that was comparable to a human observer (98% agreement) over 111 different short videos (total 284 min) at a resolution of 1 s. To test the system over longer experimental conditions, the responses of two inbred strains, C57BL/6NJ and C57BL/6J, were recorded over 90 min post formalin challenge. The automated system easily scored over 80 h of video and revealed strain differences in both response timing and amplitude. This machine learning scoring system provides the required accuracy, consistency, and ease of use that could make the formalin assay a feasible choice for large-scale genetic studies.


Assuntos
Comportamento Animal/efeitos dos fármacos , Aprendizado de Máquina , Nociceptividade/efeitos dos fármacos , Gravação em Vídeo/métodos , Algoritmos , Animais , Automação , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reprodutibilidade dos Testes
14.
Blood ; 132(24): e35-e46, 2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-30275110

RESUMO

Antithrombotic therapies reduce cardiovascular diseases by preventing arterial thrombosis and thromboembolism, but at expense of increased bleeding risks. Arterial thrombosis studies using genetically modified mice have been invaluable for identification of new molecular targets. Because of low sample sizes and heterogeneity in approaches or methodologies, a formal meta-analysis to compare studies of mice with single-gene defects encountered major limitations. To overcome these, we developed a novel synthesis approach to quantitatively scale 1514 published studies of arterial thrombus formation (in vivo and in vitro), thromboembolism, and tail-bleeding of genetically modified mice. Using a newly defined consistency parameter (CP), indicating the strength of published data, comparisons were made of 431 mouse genes, of which 17 consistently contributed to thrombus formation without affecting hemostasis. Ranking analysis indicated high correlations between collagen-dependent thrombosis models in vivo (FeCl3 injury or ligation/compression) and in vitro. Integration of scores and CP values resulted in a network of protein interactions in thrombosis and hemostasis (PITH), which was combined with databases of genetically linked human bleeding and thrombotic disorders. The network contained 2946 nodes linked to modifying genes of thrombus formation, mostly with expression in megakaryocytes. Reactome pathway analysis and network characteristics revealed multiple novel genes with potential contribution to thrombosis/hemostasis. Studies with additional knockout mice revealed that 4 of 8 (Apoe, Fpr2, Ifnar1, Vps13a) new genes were modifying in thrombus formation. The PITH network further: (i) revealed a high similarity of murine and human hemostatic and thrombotic processes and (ii) identified multiple new candidate proteins regulating these processes.


Assuntos
Hemorragia , Trombose , Animais , Modelos Animais de Doenças , Hemorragia/genética , Hemorragia/metabolismo , Hemorragia/patologia , Humanos , Camundongos , Camundongos Knockout , Trombose/genética , Trombose/metabolismo , Trombose/patologia
15.
Hum Mol Genet ; 25(2): 291-307, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26604141

RESUMO

Zinc finger motifs are distributed amongst many eukaryotic protein families, directing nucleic acid-protein and protein-protein interactions. Zinc finger protein 106 (ZFP106) has previously been associated with roles in immune response, muscle differentiation, testes development and DNA damage, although little is known about its specific function. To further investigate the function of ZFP106, we performed an in-depth characterization of Zfp106 deficient mice (Zfp106(-/-)), and we report a novel role for ZFP106 in motor and sensory neuronal maintenance and survival. Zfp106(-/-) mice develop severe motor abnormalities, major deficits in muscle strength and histopathological changes in muscle. Intriguingly, despite being highly expressed throughout the central nervous system, Zfp106(-/-) mice undergo selective motor and sensory neuronal and axonal degeneration specific to the spinal cord and peripheral nervous system. Neurodegeneration does not occur during development of Zfp106(-/-) mice, suggesting that ZFP106 is likely required for the maintenance of mature peripheral motor and sensory neurons. Analysis of embryonic Zfp106(-/-) motor neurons revealed deficits in mitochondrial function, with an inhibition of Complex I within the mitochondrial electron transport chain. Our results highlight a vital role for ZFP106 in sensory and motor neuron maintenance and reveal a novel player in mitochondrial dysfunction and neurodegeneration.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neurônios Motores/metabolismo , Doenças Neurodegenerativas/genética , Células Receptoras Sensoriais/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Neurônios Motores/fisiologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Células Receptoras Sensoriais/fisiologia
16.
PLoS Biol ; 13(5): e1002151, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25992600

RESUMO

The Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines were developed to address the lack of reproducibility in biomedical animal studies and improve the communication of research findings. While intended to guide the preparation of peer-reviewed manuscripts, the principles of transparent reporting are also fundamental for in vivo databases. Here, we describe the benefits and challenges of applying the guidelines for the International Mouse Phenotyping Consortium (IMPC), whose goal is to produce and phenotype 20,000 knockout mouse strains in a reproducible manner across ten research centres. In addition to ensuring the transparency and reproducibility of the IMPC, the solutions to the challenges of applying the ARRIVE guidelines in the context of IMPC will provide a resource to help guide similar initiatives in the future.


Assuntos
Experimentação Animal/normas , Bases de Dados como Assunto , Guias como Assunto , Fenótipo , Animais , Camundongos
17.
PLoS Genet ; 10(10): e1004688, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25356849

RESUMO

Spinster homolog 2 (Spns2) acts as a Sphingosine-1-phosphate (S1P) transporter in zebrafish and mice, regulating heart development and lymphocyte trafficking respectively. S1P is a biologically active lysophospholipid with multiple roles in signalling. The mechanism of action of Spns2 is still elusive in mammals. Here, we report that Spns2-deficient mice rapidly lost auditory sensitivity and endocochlear potential (EP) from 2 to 3 weeks old. We found progressive degeneration of sensory hair cells in the organ of Corti, but the earliest defect was a decline in the EP, suggesting that dysfunction of the lateral wall was the primary lesion. In the lateral wall of adult mutants, we observed structural changes of marginal cell boundaries and of strial capillaries, and reduced expression of several key proteins involved in the generation of the EP (Kcnj10, Kcnq1, Gjb2 and Gjb6), but these changes were likely to be secondary. Permeability of the boundaries of the stria vascularis and of the strial capillaries appeared normal. We also found focal retinal degeneration and anomalies of retinal capillaries together with anterior eye defects in Spns2 mutant mice. Targeted inactivation of Spns2 in red blood cells, platelets, or lymphatic or vascular endothelial cells did not affect hearing, but targeted ablation of Spns2 in the cochlea using a Sox10-Cre allele produced a similar auditory phenotype to the original mutation, suggesting that local Spns2 expression is critical for hearing in mammals. These findings indicate that Spns2 is required for normal maintenance of the EP and hence for normal auditory function, and support a role for S1P signalling in hearing.


Assuntos
Proteínas de Transporte de Ânions/genética , Cóclea/patologia , Orelha Interna/patologia , Perda Auditiva/genética , Idade de Início , Animais , Proteínas de Transporte de Ânions/deficiência , Proteínas de Transporte de Ânions/metabolismo , Segmento Anterior do Olho/metabolismo , Segmento Anterior do Olho/patologia , Cóclea/metabolismo , Conexina 26 , Conexinas , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patologia , Perda Auditiva/metabolismo , Perda Auditiva/patologia , Lisofosfolipídeos/metabolismo , Camundongos , Organogênese/genética , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Estria Vascular/patologia , Peixe-Zebra
18.
PLoS Genet ; 10(10): e1004705, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25340873

RESUMO

The skin is a highly regenerative organ which plays critical roles in protecting the body and sensing its environment. Consequently, morbidity and mortality associated with skin defects represent a significant health issue. To identify genes important in skin development and homeostasis, we have applied a high throughput, multi-parameter phenotype screen to the conditional targeted mutant mice generated by the Wellcome Trust Sanger Institute's Mouse Genetics Project (Sanger-MGP). A total of 562 different mouse lines were subjected to a variety of tests assessing cutaneous expression, macroscopic clinical disease, histological change, hair follicle cycling, and aberrant marker expression. Cutaneous lesions were associated with mutations in 23 different genes. Many of these were not previously associated with skin disease in the organ (Mysm1, Vangl1, Trpc4ap, Nom1, Sparc, Farp2, and Prkab1), while others were ascribed new cutaneous functions on the basis of the screening approach (Krt76, Lrig1, Myo5a, Nsun2, and Nf1). The integration of these skin specific screening protocols into the Sanger-MGP primary phenotyping pipelines marks the largest reported reverse genetic screen undertaken in any organ and defines approaches to maximise the productivity of future projects of this nature, while flagging genes for further characterisation.


Assuntos
Mutação/genética , Fenótipo , Fenômenos Fisiológicos da Pele/genética , Animais , Células-Tronco Embrionárias , Folículo Piloso/metabolismo , Folículo Piloso/fisiologia , Camundongos , Genética Reversa
19.
FASEB J ; 29(5): 1676-87, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25526730

RESUMO

The importance of epigenetic changes in the development of hepatic steatosis is largely unknown. The histone variant macroH2A1 under alternative splicing gives rise to macroH2A1.1 and macroH2A1.2. In this study, we show that the macroH2A1 isoforms play an important role in the regulation of lipid accumulation in hepatocytes. Hepatoma cell line and immortalized human hepatocytes transiently transfected or knocked down with macroH2A1 isoforms were used as in vitro model of fat-induced steatosis. Gene expressions were analyzed by quantitative PCR array and Western blot. Chromatin immunoprecipitation analysis was performed to check the association of histone H3 lysine 27 trimethylation (H3K27me3) and histone H3 lysine 4 trimethylation (H3K4me3) with the promoter of lipogenic genes. Livers from knockout mice that are resistant to lipid deposition despite a high-fat diet were used for histopathology. We found that macroH2A1.2 is regulated by fat uptake and that its overexpression caused an increase in lipid uptake, triglycerides, and lipogenic genes compared with macroH2A1.1. This suggests that macroH2A1.2 is important for lipid uptake, whereas macroH2A1.1 was found to be protective. The result was supported by a high positivity for macroH2A1.1 in knockout mice for genes targeted by macroH2A1 (Atp5a1 and Fam73b), that under a high-fat diet presented minimal lipidosis. Moreover, macroH2A1 isoforms differentially regulate the expression of lipogenic genes by modulating the association of the active (H3K4me3) and repressive (H3K27me3) histone marks on their promoters. This study underlines the importance of the replacement of noncanonical histones in the regulation of genes involved in lipid metabolism in the progression of steatosis.


Assuntos
Biomarcadores/metabolismo , Carcinoma Hepatocelular/patologia , Dieta Hiperlipídica/efeitos adversos , Epigenômica , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Hepatócitos/patologia , Histonas/metabolismo , Animais , Western Blotting , Carcinoma Hepatocelular/metabolismo , Células Cultivadas , Imunoprecipitação da Cromatina , Fígado Gorduroso/etiologia , Feminino , Imunofluorescência , Perfilação da Expressão Gênica , Hepatócitos/metabolismo , Histonas/genética , Humanos , Técnicas Imunoenzimáticas , Peroxidação de Lipídeos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , ATPases Mitocondriais Próton-Translocadoras/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
20.
Am J Hum Genet ; 91(6): 998-1010, 2012 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-23200864

RESUMO

Ubiquitination plays a crucial role in neurodevelopment as exemplified by Angelman syndrome, which is caused by genetic alterations of the ubiquitin ligase-encoding UBE3A gene. Although the function of UBE3A has been widely studied, little is known about its paralog UBE3B. By using exome and capillary sequencing, we here identify biallelic UBE3B mutations in four patients from three unrelated families presenting an autosomal-recessive blepharophimosis-ptosis-intellectual-disability syndrome characterized by developmental delay, growth retardation with a small head circumference, facial dysmorphisms, and low cholesterol levels. UBE3B encodes an uncharacterized E3 ubiquitin ligase. The identified UBE3B variants include one frameshift and two splice-site mutations as well as a missense substitution affecting the highly conserved HECT domain. Disruption of mouse Ube3b leads to reduced viability and recapitulates key aspects of the human disorder, such as reduced weight and brain size and a downregulation of cholesterol synthesis. We establish that the probable Caenorhabditis elegans ortholog of UBE3B, oxi-1, functions in the ubiquitin/proteasome system in vivo and is especially required under oxidative stress conditions. Our data reveal the pleiotropic effects of UBE3B deficiency and reinforce the physiological importance of ubiquitination in neuronal development and function in mammals.


Assuntos
Blefarofimose/genética , Blefaroptose/genética , Deficiência Intelectual/genética , Ubiquitina-Proteína Ligases/genética , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Blefarofimose/diagnóstico , Blefaroptose/diagnóstico , Encéfalo/patologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Sistema Nervoso Central , Criança , Pré-Escolar , Exoma , Fácies , Feminino , Genótipo , Humanos , Lactente , Deficiência Intelectual/diagnóstico , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Knockout , Mutação , Estresse Oxidativo , Síndrome , Ubiquitina-Proteína Ligases/deficiência
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