RESUMEN
Schizophrenia is highly heritable, yet its underlying pathophysiology remains largely unknown. Among the most well-replicated findings in neurobiological studies of schizophrenia are deficits in myelination and white matter integrity; however, direct etiological genetic and cellular evidence has thus far been lacking. Here, we implement a family-based approach for genetic discovery in schizophrenia combined with functional analysis using induced pluripotent stem cells (iPSCs). We observed familial segregation of two rare missense mutations in Chondroitin Sulfate Proteoglycan 4 (CSPG4) (c.391G > A [p.A131T], MAF 7.79 × 10-5 and c.2702T > G [p.V901G], MAF 2.51 × 10-3). The CSPG4A131T mutation was absent from the Swedish Schizophrenia Exome Sequencing Study (2536 cases, 2543 controls), while the CSPG4V901G mutation was nominally enriched in cases (11 cases vs. 3 controls, P = 0.026, OR 3.77, 95% CI 1.05-13.52). CSPG4/NG2 is a hallmark protein of oligodendrocyte progenitor cells (OPCs). iPSC-derived OPCs from CSPG4A131T mutation carriers exhibited abnormal post-translational processing (P = 0.029), subcellular localization of mutant NG2 (P = 0.007), as well as aberrant cellular morphology (P = 3.0 × 10-8), viability (P = 8.9 × 10-7), and myelination potential (P = 0.038). Moreover, transfection of healthy non-carrier sibling OPCs confirmed a pathogenic effect on cell survival of both the CSPG4A131T (P = 0.006) and CSPG4V901G (P = 3.4 × 10-4) mutations. Finally, in vivo diffusion tensor imaging of CSPG4A131T mutation carriers demonstrated a reduction of brain white matter integrity compared to unaffected sibling and matched general population controls (P = 2.2 × 10-5). Together, our findings provide a convergence of genetic and functional evidence to implicate OPC dysfunction as a candidate pathophysiological mechanism of familial schizophrenia.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/genética , Proteínas de la Membrana/genética , Células Precursoras de Oligodendrocitos/metabolismo , Esquizofrenia/genética , Adulto , Antígenos/genética , Diferenciación Celular/fisiología , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Imagen de Difusión Tensora , Familia , Femenino , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Mutación/genética , Células Precursoras de Oligodendrocitos/fisiología , Oligodendroglía/metabolismo , Linaje , Proteoglicanos/genética , Esquizofrenia/metabolismo , Sustancia Blanca/metabolismoAsunto(s)
Mutación , Regiones Promotoras Genéticas/genética , Globinas beta/genética , Talasemia beta/genética , Animales , Silenciador del Gen , Humanos , Ratones , Ratones Transgénicos , Motivos de Nucleótidos , Unión Proteica , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
BACKGROUND: The zinc finger protein Sp2 (specificity protein 2) is a member of the glutamine-rich Sp family of transcription factors. Despite its close similarity to Sp1, Sp3 and Sp4, Sp2 does not bind to DNA or activate transcription when expressed in mammalian cell lines. The expression pattern and the biological relevance of Sp2 in the mouse are unknown. METHODOLOGY/PRINCIPAL FINDINGS: Whole-mount in situ hybridization of mouse embryos between E7.5 and E9.5 revealed abundant expression in most embryonic and extra-embryonic tissues. In order to unravel the biological relevance of Sp2, we have targeted the Sp2 gene by a tri-loxP strategy. Constitutive Sp2null and conditional Sp2cko knockout alleles were obtained by crossings with appropriate Cre recombinase expressing mice. Constitutive disruption of the mouse Sp2 gene (Sp2null) resulted in severe growth retardation and lethality before E9.5. Mouse embryonic fibroblasts (MEFs) derived from Sp2null embryos at E9.5 failed to grow. Cre-mediated ablation of Sp2 in Sp2cko/cko MEFs obtained from E13.5 strongly impaired cell proliferation. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that Sp2 is essential for early mouse development and autonomous proliferation of MEFs in culture. Comparison of the Sp2 knockout phenotype with the phenotypes of Sp1, Sp3 and Sp4 knockout strains shows that, despite their structural similarity and evolutionary relationship, all four glutamine-rich members of the Sp family of transcription factors have distinct non-redundant functions in vivo.
Asunto(s)
Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Factor de Transcripción Sp2/fisiología , Animales , Proliferación Celular , ADN/metabolismo , Evolución Molecular , Femenino , Hibridación in Situ , Masculino , Ratones , Ratones Noqueados , Fenotipo , Factores de Tiempo , Transcripción GenéticaRESUMEN
Germ line gene transposition technology has been used to generate "libraries" of flies and worms carrying genomewide mutations. Phenotypic screening and DNA sequencing of such libraries provide functional information resulting from insertional events in target genes. There is also a great need to have a fast and efficient way to generate mouse mutants in vivo to model developmental defects and human diseases. Here we describe an optimized mammalian germ line transposition system active during early mouse spermatogenesis using the Minos transposon. Transposon-positive progeny carry on average more than 2 new transpositions, and 45 to 100% of the progeny carry an insertion in a gene. The optimized Minos-based system was tested in a small rapid dominant functional screen to identify mutated genes likely to cause measurable cardiovascular "disease" phenotypes in progeny/embryos. Importantly this system allows rapid screening for modifier genes.
Asunto(s)
Elementos Transponibles de ADN , Células Germinativas , Ratones Mutantes/genética , Ratones Transgénicos/genética , Mutagénesis , Transposasas/genética , Animales , Enfermedades Cardiovasculares/embriología , Enfermedades Cardiovasculares/genética , Técnicas de Sustitución del Gen , Ratones , Ratones Endogámicos C57BL , Espermatogénesis/genéticaRESUMEN
Locus control regions (LCRs) are defined by their ability to confer reproducible physiological levels of transgene expression in mice and therefore thought to possess the ability to generate dominantly a transcriptionally active chromatin structure. We report the first characterization of a muscle-cell-specific LCR, which is linked to the human desmin gene (DES). The DES LCR consists of five regions of muscle-specific DNase I hypersensitivity (HS) localized between -9 and -18 kb 5' of DES and reproducibly drives full physiological levels of expression in all muscle cell types. The DES LCR DNase I HS regions are highly conserved between humans and other mammals and can potentially bind a broad range of muscle-specific and ubiquitous transcription factors. Bioinformatics and direct molecular analysis show that the DES locus consists of three muscle-specific (DES) or muscle preferentially expressed genes (APEG1 and SPEG, the human orthologue of murine striated-muscle-specific serine/threonine protein kinase, Speg). The DES LCR may therefore regulate expression of SPEG and APEG1 as well as DES.
Asunto(s)
Desmina/genética , Región de Control de Posición , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Secuencia Conservada , ADN/genética , ADN/metabolismo , Desoxirribonucleasa I , Expresión Génica , Ligamiento Genético , Humanos , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Proteínas Musculares/genética , Músculos/metabolismo , Proteínas Serina-Treonina Quinasas , Ratas , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , Transcripción GenéticaRESUMEN
The presence of porcine endogenous retroviruses presents a potential risk of transmission of infectious diseases (xenozoonosis) if tissues and organs from genetically modified pigs are to be used in xenotransplantation. Here, we report that intracellular expression of a llama single-domain antibody against p15, the matrix domain protein of the porcine endogenous retrovirus Gag polyprotein, blocks retrovirus production, providing the possibility of eliminating the risk of infection in xenotransplantation.
Asunto(s)
Anticuerpos Antivirales/fisiología , Retrovirus Endógenos/fisiología , Productos del Gen gag/antagonistas & inhibidores , Infecciones por Retroviridae/prevención & control , Porcinos/virología , Trasplante Heterólogo/efectos adversos , Proteínas de la Matriz Viral/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Afinidad de Anticuerpos , Mapeo Epitopo , Productos del Gen gag/inmunología , Humanos , Sueros Inmunes/inmunología , Cadenas Pesadas de Inmunoglobulina/fisiología , Región Variable de Inmunoglobulina/fisiología , Datos de Secuencia Molecular , Proteínas de la Matriz Viral/inmunología , Replicación ViralRESUMEN
Double-stranded RNA (dsRNA) has been shown to interfere with the function of specific genes in various invertebrate species. The application of dsRNA interference (RNAi) in vertebrates (zebrafish and mouse) is still limited to embryos and it is not clear whether the method is generally applicable. Using a transgenic mouse model we investigated whether a stably inherited dsRNA introduced as a transgene can interfere with the expression of a specific target gene in erythroid tissue during development. In our globin gene system we do not observe any specific RNA interference. We, therefore, also introduced another gene that may be involved in a mechanism of post transcriptional gene silencing (PTGS), namely RNA-dependent RNA polymerase (RdRP) that was proposed to be involved in producing RNAs that trigger PTGS in plants. However, even though the tomato RdRP is catalytically active in erythroid tissue, no RNAi was observed.
Asunto(s)
Interferencia de ARN , ARN Polimerasa Dependiente del ARN/genética , Solanum lycopersicum/genética , Animales , Globinas/genética , Humanos , Ratones , Ratones Transgénicos , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismoRESUMEN
The Sca-1 surface glycoprotein is used routinely as a marker for haematopoietic stem cell enrichment. Two allelic genes, Ly-6A and Ly-6E, encode this marker and appear to be differentially regulated in haematopoietic cells and haematopoietic stem cells. The Sca-1 protein has been shown to be expressed at a greater frequency in these cells from Ly-6A strains of mice. To study the specific expression pattern and haematopoietic regulation of the Ly-6A gene, we constructed a 14 kb cassette from a genomic Ly-6A fragment, inserted a lacZ reporter gene and created transgenic mice. We found that the Ly-6A lacZ transgene was expressed in the haematopoietic tissues and predominantly in the T-lymphoid lineage. Some expression was also found in the B-lymphoid and myeloid lineages. We demonstrated functional haematopoietic stem cell enrichment by sorting for beta-galactosidase-expressing cells from the bone marrow. In addition, we found an interesting embryonic expression pattern in the AGM region, the site of the first haematopoietic stem cell generation. Surprisingly, when compared with data from Ly-6E lacZ transgenic mice, our results suggest that the Ly-6A cassette does not improve lacZ marker gene expression in haematopoietic cells.
Asunto(s)
Antígenos Ly/genética , Embrión de Mamíferos/metabolismo , Células Madre Hematopoyéticas/metabolismo , Proteínas de la Membrana/genética , Linfocitos T/metabolismo , Animales , Northern Blotting/métodos , Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea , Femenino , Citometría de Flujo , Expresión Génica , Ingeniería Genética , Marcadores Genéticos , Operón Lac , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones TransgénicosRESUMEN
Este libro se ha estructurado en tres partes que se interrelacionan y brindan una visión compleja del proceso de la Educación Popular. El primer título "El proceso peruano de la educación popular", ofrece una periodización global sobre el tema, así como una evolución histórica del concepto. El siguiente título sobre "Los centros en el proceso peruano de la educación popular", son reflexiones que plantean miradas históricas hacia la educación popular pero muy en función del rol de las organizacines no gubernamentales. El tercer título sobre "El proceso latinoamericano de la educación popular", nos permite un contexto más amplio para comprender las diversas influencias y diferencias del proceso latinoamericano y peruano
