RESUMEN
The RIKEN integrated database of mammals (http://scinets.org/db/mammal) is the official undertaking to integrate its mammalian databases produced from multiple large-scale programs that have been promoted by the institute. The database integrates not only RIKEN's original databases, such as FANTOM, the ENU mutagenesis program, the RIKEN Cerebellar Development Transcriptome Database and the Bioresource Database, but also imported data from public databases, such as Ensembl, MGI and biomedical ontologies. Our integrated database has been implemented on the infrastructure of publication medium for databases, termed SciNetS/SciNeS, or the Scientists' Networking System, where the data and metadata are structured as a semantic web and are downloadable in various standardized formats. The top-level ontology-based implementation of mammal-related data directly integrates the representative knowledge and individual data records in existing databases to ensure advanced cross-database searches and reduced unevenness of the data management operations. Through the development of this database, we propose a novel methodology for the development of standardized comprehensive management of heterogeneous data sets in multiple databases to improve the sustainability, accessibility, utility and publicity of the data of biomedical information.
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Bases de Datos Factuales , Bases de Datos Genéticas , Mamíferos/genética , Animales , Humanos , Internet , Mamíferos/metabolismo , Ratones , Integración de Sistemas , Interfaz Usuario-ComputadorRESUMEN
UNLABELLED: This article reports the development of SDOP-DB, which can provide definite, detailed and easy comparison of experimental protocols used in mouse phenotypic analyses among institutes or laboratories. Because SDOP-DB is fully compliant with international standards, it can act as a practical foundation for international sharing and integration of mouse phenotypic information. AVAILABILITY: SDOP-DB (http://www.brc.riken.jp/lab/bpmp/SDOP/).
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Bases de Datos Factuales , Genómica/métodos , Ratones , Fenotipo , Programas Informáticos , Animales , Internet , Interfaz Usuario-ComputadorRESUMEN
A systematic and comprehensive phenotyping platform has been developed by the RIKEN ENU-mutagenesis project between 1999 and 2007. As a result of phenotype screening on this platform, we have discovered about 400 mutants as animal models for human diseases. All information regarding these mouse mutants is now available to the public through our home page (http://www.brc.riken.jp/lab/gsc/mouse/indexJ.html). In 2008, we reconstructed the existing phenotyping platform and built a new platform. The new system has a hierarchical structure, consisting of a fundamental pipeline that utilizes the existing platform and an additional pipeline, which is optimized for more in-depth phenotyping assays. Using this system, we have started to perform more comprehensive phenotyping of mouse mutants. We have opened this system to Japanese scientists as the Japanese Mouse Clinic. It is anticipated that existing mouse mutants will be reevaluated as disease models by identifying novel phenotypes on the new platform. We will share detailed information about the standard operating procedures (SOPs) of our phenotyping analyses with other related large-scale projects, such as the European Mouse Disease Clinic (EUMODIC) and the German Mouse Clinic (GMC). Moreover, we will contribute to international efforts to standardize mouse phenotype data by sharing annotation of mutant phenotypes, which are made by internationally standardized methods, with other related projects.
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Bases de Datos Factuales , Modelos Animales de Enfermedad , Centros de Información/organización & administración , Ratones Mutantes/genética , Crianza de Animales Domésticos , Animales , Femenino , Genoma , Humanos , Cooperación Internacional , Masculino , Ratones , Ratones Endogámicos , Fenotipo , Estándares de ReferenciaRESUMEN
Although repetitive elements pervade mammalian genomes, their overall contribution to transcriptional activity is poorly defined. Here, as part of the FANTOM4 project, we report that 6-30% of cap-selected mouse and human RNA transcripts initiate within repetitive elements. Analysis of approximately 250,000 retrotransposon-derived transcription start sites shows that the associated transcripts are generally tissue specific, coincide with gene-dense regions and form pronounced clusters when aligned to full-length retrotransposon sequences. Retrotransposons located immediately 5' of protein-coding loci frequently function as alternative promoters and/or express noncoding RNAs. More than a quarter of RefSeqs possess a retrotransposon in their 3' UTR, with strong evidence for the reduced expression of these transcripts relative to retrotransposon-free transcripts. Finally, a genome-wide screen identifies 23,000 candidate regulatory regions derived from retrotransposons, in addition to more than 2,000 examples of bidirectional transcription. We conclude that retrotransposon transcription has a key influence upon the transcriptional output of the mammalian genome.
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Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Retroelementos/genética , Regiones no Traducidas 3'/genética , Regiones no Traducidas 3'/metabolismo , Animales , Células Cultivadas , Humanos , Mamíferos/genética , Ratones , Regiones Promotoras Genéticas , ARN Mensajero , ARN no Traducido/metabolismoRESUMEN
BACKGROUND: The transcriptome of the cerebral cortex is remarkably homogeneous, with variations being stronger between individuals than between areas. It is thought that due to the presence of many distinct cell types, differences within one cell population will be averaged with the noise from others. Studies of sorted cells expressing the same transgene have shown that cell populations can be distinguished according to their transcriptional profile. METHODOLOGY: We have prepared a low-redundancy set of 16,209 full-length cDNA clones which represents the transcriptome of the mouse visual cortex in its coding and non-coding aspects. Using an independent tag-based approach, CAGE, we confirmed the cortical expression of 72% of the clones. Clones were amplified by PCR and spotted on glass slides, and we interrogated the microarrays with RNA from flow-sorted fluorescent cells from the cerebral cortex of parvalbumin-egfp transgenic mice. CONCLUSIONS: We provide an annotated cDNA clone collection which is particularly suitable for transcriptomic analysis in the mouse brain. Spotting it on microarrays, we compared the transcriptome of EGFP positive and negative cells in a parvalbumin-egfp transgenic background and showed that more than 30% of clones are differentially expressed. Our clone collection will be a useful resource for the study of the transcriptome of single cell types in the cerebral cortex.
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Encéfalo/fisiología , Perfilación de la Expresión Génica , Transcripción Genética , Corteza Visual/fisiología , Animales , Cerebelo/fisiología , ADN Complementario/genética , Etiquetas de Secuencia Expresada , Biblioteca de Genes , Ratones , Modelos Estadísticos , Proteínas del Tejido Nervioso/genéticaRESUMEN
Rheumatoid arthritis is a chronic inflammatory disease with a high prevalence and substantial socioeconomic burden. Despite intense research efforts, its aetiology and pathogenesis remain poorly understood. To identify novel genes and/or cellular pathways involved in the pathogenesis of the disease, we utilized a well-recognized tumour necrosis factor-driven animal model of this disease and performed high-throughput expression profiling with subtractive cDNA libraries and oligonucleotide microarray hybridizations, coupled with independent statistical analysis. This twin approach was validated by a number of different methods in other animal models of arthritis as well as in human patient samples, thus creating a unique list of disease modifiers of potential therapeutic value. Importantly, and through the integration of genetic linkage analysis and Gene Ontology-assisted functional discovery, we identified the gelsolin-driven synovial fibroblast cytoskeletal rearrangements as a novel pathophysiological determinant of the disease.
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Artritis Reumatoide/metabolismo , Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Membrana Sinovial/citología , Animales , Artritis Reumatoide/fisiopatología , Biblioteca de Genes , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Sitios de Carácter Cuantitativo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
It is becoming increasingly clear that alternative splicing enables the complex development and homeostasis of higher organisms. To gain a better understanding of how splicing contributes to regulatory pathways, we have developed an alternative splicing library approach for the identification of alternatively spliced exons and their flanking regions by alternative splicing sequence enriched tags sequencing. Here, we have applied our approach to mouse melan-c melanocyte and B16-F10Y melanoma cell lines, in which 5,401 genes were found to be alternatively spliced. These genes include those encoding important regulatory factors such as cyclin D2, Ilk, MAPK12, MAPK14, RAB4, melastatin 1 and previously unidentified splicing events for 436 genes. Real-time PCR further identified cell line-specific exons for Tmc6, Abi1, Sorbs1, Ndel1 and Snx16. Thus, the ASL approach proved effective in identifying splicing events, which suggest that alternative splicing is important in melanoma development.
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Empalme Alternativo/genética , Biomarcadores de Tumor/genética , Clonación Molecular/métodos , Exones/genética , Melanoma/genética , Biblioteca de Péptidos , Análisis de Secuencia de ADN/métodos , Animales , Secuencia de Bases , Línea Celular Tumoral , Melanocitos/fisiología , Ratones , Datos de Secuencia Molecular , ARN Mensajero/genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Alineación de Secuencia/métodosRESUMEN
We introduce cap analysis gene expression (CAGE), which is based on preparation and sequencing of concatamers of DNA tags deriving from the initial 20 nucleotides from 5' end mRNAs. CAGE allows high-throughout gene expression analysis and the profiling of transcriptional start points (TSP), including promoter usage analysis. By analyzing four libraries (brain, cortex, hippocampus, and cerebellum), we redefined more accurately the TSPs of 11-27% of the analyzed transcriptional units that were hit. The frequency of CAGE tags correlates well with results from other analyses, such as serial analysis of gene expression, and furthermore maps the TSPs more accurately, including in tissue-specific cases. The high-throughput nature of this technology paves the way for understanding gene networks via correlation of promoter usage and gene transcriptional factor expression.
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Regulación de la Expresión Génica , Regiones Promotoras Genéticas/genética , Transcripción Genética/genética , Animales , Secuencia de Bases , Química Encefálica , Carboxipeptidasa H/genética , ADN/química , ADN/genética , Técnicas Genéticas , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Reproducibilidad de los ResultadosRESUMEN
The normalization and subtraction of highly expressed cDNAs from relatively large tissues before cloning dramatically enhanced the gene discovery by sequencing for the mouse full-length cDNA encyclopedia, but these methods have not been suitable for limited RNA materials. To normalize and subtract full-length cDNA libraries derived from limited quantities of total RNA, here we report a method to subtract plasmid libraries excised from size-unbiased amplified lambda phage cDNA libraries that avoids heavily biasing steps such as PCR and plasmid library amplification. The proportion of full-length cDNAs and the gene discovery rate are high, and library diversity can be validated by in silico randomization.
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Perfilación de la Expresión Génica/métodos , Biblioteca de Genes , Alineación de Secuencia/métodos , Análisis de Secuencia de ADN/métodos , Transcripción Genética/genética , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
We report the construction of the mouse full-length cDNA encyclopedia,the most extensive view of a complex transcriptome,on the basis of preparing and sequencing 246 libraries. Before cloning,cDNAs were enriched in full-length by Cap-Trapper,and in most cases,aggressively subtracted/normalized. We have produced 1,442,236 successful 3'-end sequences clustered into 171,144 groups, from which 60,770 clones were fully sequenced cDNAs annotated in the FANTOM-2 annotation. We have also produced 547,149 5' end reads,which clustered into 124,258 groups. Altogether, these cDNAs were further grouped in 70,000 transcriptional units (TU),which represent the best coverage of a transcriptome so far. By monitoring the extent of normalization/subtraction, we define the tentative equivalent coverage (TEC),which was estimated to be equivalent to >12,000,000 ESTs derived from standard libraries. High coverage explains discrepancies between the very large numbers of clusters (and TUs) of this project,which also include non-protein-coding RNAs,and the lower gene number estimation of genome annotations. Altogether,5'-end clusters identify regions that are potential promoters for 8637 known genes and 5'-end clusters suggest the presence of almost 63,000 transcriptional starting points. An estimate of the frequency of polyadenylation signals suggests that at least half of the singletons in the EST set represent real mRNAs. Clones accounting for about half of the predicted TUs await further sequencing. The continued high-discovery rate suggests that the task of transcriptome discovery is not yet complete.
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Clonación Molecular , ADN Complementario/genética , Genoma , Transcripción Genética/genética , Animales , Clonación Molecular/métodos , Análisis por Conglomerados , Bases de Datos Genéticas/estadística & datos numéricos , Etiquetas de Secuencia Expresada , Perfilación de la Expresión Génica/métodos , Perfilación de la Expresión Génica/estadística & datos numéricos , Biblioteca de Genes , Genes/genética , Genes/fisiología , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos/genética , Poliadenilación/genética , Caperuzas de ARN/genética , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de ADN/estadística & datos numéricosRESUMEN
To determine the chromosomal positions of expressed rice genes, we have performed an expressed sequence tag (EST) mapping project by polymerase chain reaction-based yeast artificial chromosome (YAC) screening. Specific primers designed from 6713 unique EST sequences derived from 19 cDNA libraries were screened on 4387 YAC clones and used for map construction in combination with genetic analysis. Here, we describe the establishment of a comprehensive YAC-based rice transcript map that contains 6591 EST sites and covers 80.8% of the rice genome. Chromosomes 1, 2, and 3 have relatively high EST densities, approximately twice those of chromosomes 11 and 12, and contain 41% of the total EST sites on the map. Most of the EST-dense regions are distributed on the distal regions of each chromosome arm. Genomic regions flanking the centromeres for most of the chromosomes have lower EST density. Recombination frequency in these regions is suppressed significantly. Our EST mapping also shows that 40% of the assigned ESTs occupy only approximately 21% of the entire genome. The rice transcript map has been a valuable resource for genetic study, gene isolation, and genome sequencing at the Rice Genome Research Program and should become an important tool for comparative analysis of chromosome structure and evolution among the cereals.