Visualizing the Spatial Relationship of the Genome with the Nuclear Envelope Using Fluorescence In Situ Hybridization
| Autor: | Mai Hassan Ahmed, Ural Bikkul, Joanna M. Bridger, Helen A. Foster, Lauren S. Godwin, Craig S. Clements |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Genome gene positioning 03 medical and health sciences 0302 clinical medicine medicine genome organization fluorescence in situ hybridization Integral membrane protein 3D-FISH Genomic organization nuclear lamins 2D-FISH Chemistry Hybridization probe Chromatin binding nuclear envelope chromosome territories Molecular biology Chromatin Cell biology Cell nucleus 030104 developmental biology medicine.anatomical_structure Nuclear lamina 030217 neurology & neurosurgery |
| Zdroj: | Methods in Molecular Biology ISBN: 9781493935284 |
| Popis: | The genome has a special relationship with the nuclear envelope in cells. Much of the genome is anchored at the nuclear periphery, tethered by chromatin binding proteins such nuclear lamins and other integral membrane proteins. Even though there are global assays such as DAM-ID or ChIP to assess what parts of the genome are associated with the nuclear envelope, it is also essential to be able to visualize regions of the genome in order to reveal their individual relationships with nuclear structures in single cells. This is executed by fluorescence in situ hybridization (FISH) in 2-dimensional flattened nuclei (2D-FISH) or 3-dimensionally preserved cells (3D-FISH) in combination with indirect immunofluorescence to reveal structural proteins. This chapter explains the protocols for 2D- and 3D-FISH in combination with indirect immunofluorescence and discusses options for image capture and analysis. Due to the nuclear envelope proteins being part of the non-extractable nucleoskeleton, we also describe how to prepare DNA halos through salt extraction and how they can be used to study genome behavior and association when combined with 2D-FISH. SPARKs children’s charity for funding CSC; Brunel University London Progeria Research Fund; The Gordon Memorial Trust; EURO-laminopathies consortium FP6. |
| Databáze: | OpenAIRE |
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