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Visualizing the Spatial Relationship of the Genome with the Nuclear Envelope Using Fluorescence In Situ Hybridization

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The Nuclear Envelope

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1411))

Abstract

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.

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Acknowledgements

We would like to thank Dr Marion Cremer for allowing us to include some of their 3D -SIM super-resolution images of chromosomes and nuclear envelope, Dr Karen Meaburn for helpful discussions and SPARKs children’s charity for funding CSC, Brunel University London Progeria Research Fund for partial funding of UB, The Gordon Memorial Trust for supporting MHA and the EURO-laminopathies consortium FP6 for supporting LSG.

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Authors and Affiliations

  1. Division of Biosciences, College of Life and Health Sciences, Brunel University London, Uxbridge, London, UB8 3PH, UK

    Craig S. Clements, Ural Bikkul, Mai Hassan Ahmed, Helen A. Foster, Lauren S. Godwin & Joanna M. Bridger

Authors
  1. Craig S. Clements
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  2. Ural Bikkul
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  3. Mai Hassan Ahmed
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  4. Helen A. Foster
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  5. Lauren S. Godwin
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  6. Joanna M. Bridger
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Corresponding author

Correspondence to Joanna M. Bridger .

Editor information

Editors and Affiliations

  1. Department of Biochemistry, University of Leicester, Leicester, United Kingdom

    Sue Shackleton

  2. Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway

    Philippe Collas

  3. Wellcome Trust Centre for Cell Biol, University of Edinburgh, Edinburgh, United Kingdom

    Eric C. Schirmer

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Clements, C.S., Bikkul, U., Ahmed, M.H., Foster, H.A., Godwin, L.S., Bridger, J.M. (2016). Visualizing the Spatial Relationship of the Genome with the Nuclear Envelope Using Fluorescence In Situ Hybridization. In: Shackleton, S., Collas, P., Schirmer, E. (eds) The Nuclear Envelope. Methods in Molecular Biology, vol 1411. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3530-7_24

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  • DOI: https://doi.org/10.1007/978-1-4939-3530-7_24

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3528-4

  • Online ISBN: 978-1-4939-3530-7

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