RESUMEN
Fusion genes are known to be key drivers of tumor growth in several types of cancer. Traditionally, detecting fusion genes has been a difficult task based on fluorescent in situ hybridization to detect chromosomal abnormalities. More recently, RNA sequencing has enabled an increased pace of fusion gene identification. However, RNA-Seq is inefficient for the identification of fusion genes due to the high number of sequencing reads needed to detect the small number of fusion transcripts present in cells of interest. Here we describe a method, Single Primer Enrichment Technology (SPET), for targeted RNA sequencing that is customizable to any target genes, is simple to use, and efficiently detects gene fusions. Using SPET to target 5701 exons of 401 known cancer fusion genes for sequencing, we were able to identify known and previously unreported gene fusions from both fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue RNA in both normal tissue and cancer cells.
Asunto(s)
Fusión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Hibridación Fluorescente in Situ , ARN , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patología , Humanos , Fusión de Oncogenes , ARN/genéticaRESUMEN
Elucidating the molecular changes that arise during neural differentiation and fate specification is crucial for building accurate in vitro models of neurodegenerative diseases using human embryonic stem cells (hESCs). Here we review the importance of hESCs and derived progenitors in treating and modeling neurological diseases, and summarize the current efforts for the differentiation of hESCs into neural progenitors and defined neurons. We recapitulate the recent findings and discuss open questions on aspects of molecular control of gene expression by chromatin modification and methylation, posttranscriptional regulation by alternative splicing and the action of microRNAs, and protein modification. An integrative view of the different levels will hopefully provide much needed insight into understanding stem cell biology.
Asunto(s)
Células Madre Embrionarias/fisiología , Regulación del Desarrollo de la Expresión Génica , Enfermedades Neurodegenerativas/terapia , Neuronas/fisiología , Animales , Diferenciación Celular , Proliferación Celular , Tratamiento Basado en Trasplante de Células y Tejidos , Cromatina/genética , Cromatina/metabolismo , Células Madre Embrionarias/trasplante , Epigénesis Genética , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Modelos Biológicos , Neuronas/trasplanteRESUMEN
Olfactory neurons project their axons to spatially invariant glomeruli in the olfactory bulb, forming an ordered pattern of innervation comprising the olfactory sensory map. A mirror symmetry exists within this map, such that neurons expressing a given receptor typically project to one glomerulus on the medial face and one glomerulus on the lateral face of the bulb. The mechanisms underlying an olfactory neuron's choice to project medially versus laterally remain largely unknown, however. Here we demonstrate that insulin-like growth factor (IGF) signaling is required for sensory innervation of the lateral olfactory bulb. Mutations that eliminate IGF signaling cause axons destined for targets in the lateral bulb to shift to ectopic sites on the ventral-medial surface. Using primary cultures of olfactory and cerebellar neurons, we further show that IGF is a chemoattractant for axon growth cones. Together these observations reveal a role of IGF signaling in sensory map formation and axon guidance.
Asunto(s)
Axones/fisiología , Factor II del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Vías Olfatorias/fisiología , Neuronas Receptoras Olfatorias/citología , Transducción de Señal/fisiología , Animales , Axones/efectos de los fármacos , Cerebelo/citología , Factores Quimiotácticos/farmacología , Cromonas/farmacología , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Regulación del Desarrollo de la Expresión Génica/fisiología , Técnicas In Vitro , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/farmacología , Factor II del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/farmacología , Ratones , Ratones Transgénicos , Morfolinas/farmacología , Mutación/fisiología , Bulbo Olfatorio/citología , Bulbo Olfatorio/embriología , Bulbo Olfatorio/crecimiento & desarrollo , Proteína Marcadora Olfativa/genética , Proteína Marcadora Olfativa/metabolismo , Vías Olfatorias/embriología , Neuronas Receptoras Olfatorias/efectos de los fármacos , Neuronas Receptoras Olfatorias/fisiologíaRESUMEN
How olfactory sensory neurons converge on spatially invariant glomeruli in the olfactory bulb is largely unknown. In one model, olfactory sensory neurons interact with spatially restricted guidance cues in the bulb that orient and guide them to their target. Identifying differentially expressed molecules in the olfactory bulb has been extremely difficult, however, hindering a molecular analysis of convergence. Here, we describe several such genes that have been identified in a screen that compiled microarray data to create a three-dimensional model of gene expression within the mouse olfactory bulb. The expression patterns of these identified genes form the basis of a nascent spatial map of differential gene expression in the bulb.