Video Experimental Relacionado
Updated: May 8, 2026
11:03
Massively Parallel Reporter Assays in Cultured Mammalian Cells
Published on: August 17, 2014
Efectos de la posición de la cromatina analizados por miles de reporteros integrados en paralelo
Waseem Akhtar1, Johann de Jong, Alexey V Pindyurin
1Division of Molecular Genetics, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands.
Cell
|August 20, 2013
Resumen
Este estudio introduce un ensayo de reportero de alto rendimiento para medir la expresión génica. El ensayo revela cómo la estructura de la cromatina, como los dominios asociados a la lámina, impacta la regulación génica y la actividad del potenciador.
Área de la Ciencia:
- Biología Molecular Biología Molecular
- La genómica es la genómica.
- La epigenética es la epigenética.
Sus antecedentes:
- Los ensayos de genes reporteros son cruciales para el estudio de la expresión génica.
- Los ensayos de reporteros actuales tienen un rendimiento limitado, lo que dificulta el análisis a gran escala.
- Comprender la influencia de la ubicación genómica y el contexto de la cromatina en la expresión génica es esencial.
Objetivo del estudio:
- Desarrollar un ensayo reporter multiplexado de alto rendimiento para el monitoreo paralelo de la actividad transcripcional.
- Investigar el impacto del contexto local de la cromatina y los elementos reguladores en la expresión génica.
- Para analizar grandes conjuntos de datos de integraciones de reporteros para obtener nuevos conocimientos sobre la regulación génica.
Principales métodos:
- Desarrollo de un enfoque de multiplexación para el monitoreo paralelo de miles de integraciones de reporteros.
- Integración de más de 27.000 distintos reporteros construidos en células madre embrionarias de ratón.
- Análisis de los niveles de expresión del reportero en relación con la ubicación genómica y las características de la cromatina.
Principales resultados:
- Demostró una variación de aproximadamente 1.000 veces en los niveles de expresión a través de miles de integraciones de reporteros.
- Identificó dominios asociados a la lámina como atenuadores de la transcripción.
- La compactación de la cromatina mostrada es predictiva de la actividad del reportero y la influencia estimada del potenciador durante aproximadamente 20 kb.
Conclusiones:
- El ensayo de reporter múltiple permite un análisis de alto rendimiento de la regulación génica.
- La estructura de la cromatina influye significativamente en los niveles de expresión génica.
- El ensayo proporciona una plataforma flexible para diversos estudios de regulación génica.
Videos de Conceptos Relacionados
02:35Chromatin Position Affects Gene Expression
Chromatin is the massive complex of DNA and proteins packaged inside the nucleus. The complexity of chromatin folding and how it is packaged inside the nucleus greatly influences access to genetic information. Generally, the nucleus' periphery is considered transcriptionally repressive, while the cell's interior is considered a transcriptionally active area.
Topologically Associated Domains (TADs)
The 3-dimensional positioning of chromatin in the nucleus influences the timing and level of...
Topologically Associated Domains (TADs)
The 3-dimensional positioning of chromatin in the nucleus influences the timing and level of...
02:32Position-effect Variegation
In 1928, a German botanist Emil Heitz observed the moss nuclei with a DNA binding dye. He observed that while some chromatin regions decondense and spread out in the interphase nucleus, others do not. He termed them euchromatin and heterochromatin, respectively. He proposed that the heterochromatin regions reflect a functionally inactive state of the genome. It was later confirmed that heterochromatin is transcriptionally repressed, and euchromatin is transcriptionally active chromatin.
02:36Chromatin Immunoprecipitation- ChIP
Chromatin immunoprecipitation, or ChIP, is an antibody-based technique used to identify sites on DNA that bind to transcription factors of interest or histone proteins. It also helps determine the type of histone modifications such as acetylation, phosphorylation, or methylation.
Types of ChIP
ChIP can be divided into two types - X-ChIP and N-ChIP. X-ChIP involves in vivo cross-linking of histones and regulatory proteins to DNA, fragmenting the DNA by sonication, and isolating the protein-DNA...
Types of ChIP
ChIP can be divided into two types - X-ChIP and N-ChIP. X-ChIP involves in vivo cross-linking of histones and regulatory proteins to DNA, fragmenting the DNA by sonication, and isolating the protein-DNA...
02:25Spreading of Chromatin Modifications
The histone proteins in the nucleosomes are post-translationally modified (PTM) to increase or decrease access to DNA. The commonly observed PTMs are methylation, acetylation, phosphorylation, and ubiquitination of lysine amino acids in the histone H3 tail region. These histone modifications have specific meaning for the cell. Hence, they are called "histone code". The protein complex involved in histone modification is termed as "reader-writer" complex.
Writers
The writer is an enzyme that can...
Writers
The writer is an enzyme that can...
02:11Reporter Genes
Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
Commonly used reporter...
Commonly used reporter...
01:32Chromatin Modification in iPS Cells
Chromatin modification alters gene expression; therefore, scientists can add histone-modifying enzymes, histone variants, and chromatin remodeling complexes to somatic cells to aid reprogramming into pluripotent stem (iPS) cells.
Compact chromatin makes reprogramming difficult. Enzymes, such as histone demethylases and acetyltransferases, are often added during reprogramming to loosen the chromatin, making the DNA more accessible to transcription factors. Molecules that inhibit histone...
Compact chromatin makes reprogramming difficult. Enzymes, such as histone demethylases and acetyltransferases, are often added during reprogramming to loosen the chromatin, making the DNA more accessible to transcription factors. Molecules that inhibit histone...