Name: 3d multicolor dna fish tool to study nuclear architecture in human primary cells
Uploaded: 2020-01-25T13:00:00
Description: Watch this Scientific Journal Video about 3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells at JoVE.com

The authors acknowledge the technical assistance of the INGM Imaging Facility (Istituto Nazionale di Genetica Molecolare &#34;Romeo ed Enrica Invernizzi&#34; (INGM), Milan, Italy), in particular C. Cordiglieri, for assistance during 3D multicolor DNA FISH images acquisition. This work has been supported by the following grants to B.B.: EPIGEN Italian flagship program, Association Fran&#231;aise contre les Myopathies (AFM-Telethon, grant nr 18754) and Giovani Ricercatori, Italian Ministry of Health (GR-2011-02349383). This work has been supported by the following grant to F.M.: Fondazione Cariplo (Bando Giovani, grant nr 2018-0321).

1. DNA probe preparation and labelling procedures with nick translation
<ol>
	<li>Purify and clean bacterial artificial chromosomes (BACs), plasmids or PCR products with specific kits (Table of Materials), resuspend in ddH2O, check by electrophoresis on an agarose gel and quantify.</li>
	<li>Perform nick translation on 1.5&#8722;2 &#181;g of DNA from step 1.1 in a final volume of 50 &#181;L by mixing all the reagents in a 0.5 mL low binding DNA tube according to Table 1.<br ...

<ol>
	<li>Cremer, T., Cremer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+territories,+nuclear+architecture+and+gene+regulation+in+mammalian+cells.">Chromosome territories, nuclear architecture and gene regulation in mammalian cells.</a> Nature Reviews: Genetics. 2 (4), 292-301 (2001).</li><li>Lieberman-Aiden, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+mapping+of+long-range+interactions+reveals+folding+principles+of+the+human+genome.">Comprehensive mapping of long-range interactions reveals folding principles of the human genome.</a> Science. 326 (5950), 289-293 (2009).</li><li>Schmitt, A. D., Hu, M., Ren, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+mapping+and+analysis+of+chromosome+architecture.">Genome-wide mapping and analysis of chromosome architecture.</a> Nature Reviews: Molecular Cell Biology. 17 (12), 743-755 (2016).</li><li>Bonev, B., Cavalli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+function+of+the+3D+genome.">Organization and function of the 3D genome.</a> Nature Reviews: Genetics. 17 (12), 772 (2016).</li><li>van Steensel, B., Furlong, E. E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+transcription+in+shaping+the+spatial+organization+of+the+genome.">The role of transcription in shaping the spatial organization of the genome.</a> Nature Reviews: Molecular Cell Biology. 20 (6), 327-337 (2019).</li><li>Rajarajan, P., Gil, S. E., Brennand, K. J., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+genome+organization+and+cognition.">Spatial genome organization and cognition.</a> Nature Reviews: Neuroscience. 17 (11), 681-691 (2016).</li><li>Pombo, A., Dillon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+genome+architecture:+players+and+mechanisms.">Three-dimensional genome architecture: players and mechanisms.</a> Nature Reviews: Molecular Cell Biology. 16 (4), 245-257 (2015).</li><li>Fanucchi, S., Shibayama, Y., Burd, S., Weinberg, M. S., Mhlanga, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+contact+permits+transcription+between+coregulated+genes.">Chromosomal contact permits transcription between coregulated genes.</a> Cell. 155 (3), 606-620 (2013).</li><li>Therizols, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+decondensation+is+sufficient+to+alter+nuclear+organization+in+embryonic+stem+cells.">Chromatin decondensation is sufficient to alter nuclear organization in embryonic stem cells.</a> Science. 346 (6214), 1238-1242 (2014).</li><li>Gonzalez-Sandoval, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perinuclear+Anchoring+of+H3K9-Methylated+Chromatin+Stabilizes+Induced+Cell+Fate+in+C.+elegans+Embryos.">Perinuclear Anchoring of H3K9-Methylated Chromatin Stabilizes Induced Cell Fate in C. elegans Embryos.</a> Cell. 163 (6), 1333-1347 (2015).</li><li>Hubner, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodeling+of+nuclear+landscapes+during+human+myelopoietic+cell+differentiation+maintains+co-aligned+active+and+inactive+nuclear+compartments.">Remodeling of nuclear landscapes during human myelopoietic cell differentiation maintains co-aligned active and inactive nuclear compartments.</a> Epigenetics Chromatin. 8, 47 (2015).</li><li>Aymard, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+mapping+of+long-range+contacts+unveils+clustering+of+DNA+double-strand+breaks+at+damaged+active+genes.">Genome-wide mapping of long-range contacts unveils clustering of DNA double-strand breaks at damaged active genes.</a> Nature Structural &amp; Molecular Biology. 24 (4), 353-361 (2017).</li><li>Sellou, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+poly(ADP-ribose)-dependent+chromatin+remodeler+Alc1+induces+local+chromatin+relaxation+upon+DNA+damage.">The poly(ADP-ribose)-dependent chromatin remodeler Alc1 induces local chromatin relaxation upon DNA damage.</a> Molecular Biology of the Cell. 27 (24), 3791-3799 (2016).</li><li>Krijger, P. H., de Laat, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+disease-associated+gene+expression+in+the+3D+genome.">Regulation of disease-associated gene expression in the 3D genome.</a> Nature Reviews: Molecular Cell Biology. 17 (12), 771-782 (2016).</li><li>Lupianez, D. G., Spielmann, M., Mundlos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+TADs:+How+Alterations+of+Chromatin+Domains+Result+in+Disease.">Breaking TADs: How Alterations of Chromatin Domains Result in Disease.</a> Trends in Genetics. 32 (4), 225-237 (2016).</li><li>Cortesi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=4q-D4Z4+chromatin+architecture+regulates+the+transcription+of+muscle+atrophic+genes+in+facioscapulohumeral+muscular+dystrophy.">4q-D4Z4 chromatin architecture regulates the transcription of muscle atrophic genes in facioscapulohumeral muscular dystrophy.</a> Genome Research. 29 (6), 883-895 (2019).</li><li>Woltering, J. M., Noordermeer, D., Leleu, M., Duboule, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+and+divergence+of+regulatory+strategies+at+Hox+Loci+and+the+origin+of+tetrapod+digits.">Conservation and divergence of regulatory strategies at Hox Loci and the origin of tetrapod digits.</a> PLoS Biology. 12 (1), 1001773 (2014).</li><li>Woltering, J. M., Duboule, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrapod+axial+evolution+and+developmental+constraints;+Empirical+underpinning+by+a+mouse+model.">Tetrapod axial evolution and developmental constraints; Empirical underpinning by a mouse model.</a> Mechanisms of Development. 138, 64-72 (2015).</li><li>de Laat, W., Dekker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3C-based+technologies+to+study+the+shape+of+the+genome.">3C-based technologies to study the shape of the genome.</a> Methods. 58 (3), 189-191 (2012).</li><li>Denker, A., de Laat, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+second+decade+of+3C+technologies:+detailed+insights+into+nuclear+organization.">The second decade of 3C technologies: detailed insights into nuclear organization.</a> Genes &amp; Development. 30 (12), 1357-1382 (2016).</li><li>Finn, E. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+Heterogeneity+and+Intrinsic+Variation+in+Spatial+Genome+Organization.">Extensive Heterogeneity and Intrinsic Variation in Spatial Genome Organization.</a> Cell. 176 (6), 1502-1515 (2019).</li><li>Zheng, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+chromatin+interactions+with+single-molecule+precision.">Multiplex chromatin interactions with single-molecule precision.</a> Nature. 566 (7745), 558-562 (2019).</li><li>Solovei, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization+(FISH)+on+tissue+cryosections.">Fluorescence in situ hybridization (FISH) on tissue cryosections.</a> Methods in Molecular Biology. 659, 71-82 (2010).</li><li>Cremer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+3D+fluorescence+in+situ+hybridization+for+imaging+interphase+chromosomes.">Multicolor 3D fluorescence in situ hybridization for imaging interphase chromosomes.</a> Methods in Molecular Biology. 463, 205-239 (2008).</li><li>Legant, W. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-density+three-dimensional+localization+microscopy+across+large+volumes.">High-density three-dimensional localization microscopy across large volumes.</a> Nature Methods. 13 (4), 359-365 (2016).</li><li>Jungmann, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+super-resolution+imaging+with+qPAINT.">Quantitative super-resolution imaging with qPAINT.</a> Nature Methods. 13 (5), 439-442 (2016).</li><li>Chen, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+imaging+of+genomic+loci+in+living+human+cells+by+an+optimized+CRISPR/Cas+system.">Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system.</a> Cell. 155 (7), 1479-1491 (2013).</li><li>Nelles, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmable+RNA+Tracking+in+Live+Cells+with+CRISPR/Cas9.">Programmable RNA Tracking in Live Cells with CRISPR/Cas9.</a> Cell. 165 (2), 488-496 (2016).</li><li>Byron, M., Hall, L. L., Lawrence, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multifaceted+FISH+approach+to+study+endogenous+RNAs+and+DNAs+in+native+nuclear+and+cell+structures.">A multifaceted FISH approach to study endogenous RNAs and DNAs in native nuclear and cell structures.</a> Current Protocol of Human Genetics. , 15 (2013).</li><li>Chaumeil, J., Augui, S., Chow, J. C., Heard, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+immunofluorescence,+RNA+fluorescent+in+situ+hybridization,+and+DNA+fluorescent+in+situ+hybridization+to+study+chromatin+changes,+transcriptional+activity,+nuclear+organization,+and+X-chromosome+inactivation.">Combined immunofluorescence, RNA fluorescent in situ hybridization, and DNA fluorescent in situ hybridization to study chromatin changes, transcriptional activity, nuclear organization, and X-chromosome inactivation.</a> Methods in Molecular Biology. 463, 297-308 (2008).</li><li>Takizawa, T., Gudla, P. R., Guo, L., Lockett, S., Misteli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allele-specific+nuclear+positioning+of+the+monoallelically+expressed+astrocyte+marker+GFAP.">Allele-specific nuclear positioning of the monoallelically expressed astrocyte marker GFAP.</a> Genes &amp; Development. 22 (4), 489-498 (2008).</li><li>Kraus, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+3D+structured+illumination+microscopy+of+nuclear+structures.">Quantitative 3D structured illumination microscopy of nuclear structures.</a> Nature Protocols. 12 (5), 1011-1028 (2017).</li><li>Solovei, I., Cremer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-FISH+on+cultured+cells+combined+with+immunostaining.">3D-FISH on cultured cells combined with immunostaining.</a> Methods in Molecular Biology. 659, 117-126 (2010).</li><li>Skinner, B. M., Johnson, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+morphologies:+their+diversity+and+functional+relevance.">Nuclear morphologies: their diversity and functional relevance.</a> Chromosoma. 126 (2), 195-212 (2017).</li><li>Schoenfelder, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pluripotent+regulatory+circuitry+connecting+promoters+to+their+long-range+interacting+elements.">The pluripotent regulatory circuitry connecting promoters to their long-range interacting elements.</a> Genome Research. 25 (4), 582-597 (2015).</li><li>Lubeck, E., Cai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+systems+biology+by+super-resolution+imaging+and+combinatorial+labeling.">Single-cell systems biology by super-resolution imaging and combinatorial labeling.</a> Nature Methods. 9 (7), 743-748 (2012).</li><li>Beliveau, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+super-resolution+imaging+of+chromosomes+and+in+situ+haplotype+visualization+using+Oligopaint+FISH+probes.">Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes.</a> Nature Communication. 6, 7147 (2015).</li><li>Beliveau, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+design+and+synthesis+platform+for+visualizing+genomes+with+Oligopaint+FISH+probes.">Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes.</a> Proceedings of the National Academy of Sciences. 109 (52), 21301-21306 (2012).</li><li>Samacoits, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computational+framework+to+study+sub-cellular+RNA+localization.">A computational framework to study sub-cellular RNA localization.</a> Nature Communication. 9 (1), 4584 (2018).</li><li>Cardozo Gizzi, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopy-Based+Chromosome+Conformation+Capture+Enables+Simultaneous+Visualization+of+Genome+Organization+and+Transcription+in+Intact+Organisms.">Microscopy-Based Chromosome Conformation Capture Enables Simultaneous Visualization of Genome Organization and Transcription in Intact Organisms.</a> Molecular Cell. 74 (1), 212-222 (2019).</li><li>Mateo, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+DNA+folding+and+RNA+in+embryos+at+single-cell+resolution.">Visualizing DNA folding and RNA in embryos at single-cell resolution.</a> Nature. 568 (7750), 49-54 (2019).</li><li>Nir, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Walking+along+chromosomes+with+super-resolution+imaging,+contact+maps,+and+integrative+modeling.">Walking along chromosomes with super-resolution imaging, contact maps, and integrative modeling.</a> PLoS Genetics. 14 (12), 1007872 (2018).</li><li>Bintu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+chromatin+tracing+reveals+domains+and+cooperative+interactions+in+single+cells.">Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells.</a> Science. 362 (6413), (2018).</li></ol>

The current method describes a step by step protocol to perform 3D multicolor DNA FISH on a wide range of human primary cells. Although DNA FISH is a technology in wide use, 3D multicolor DNA FISH on preserved 3D interphase nuclei is still difficult to perform in many laboratories, mainly due to the characteristics of the samples used23,24.
Probe nick translation is a fundamental step for successful 3D multicolor DNA FISH; many differe...

Higher eukaryotes need to systematically condense and compact a huge amount of genetic information in the minute 3D space of the nucleus1,2,3,4. Today, we know that the genome is spatially ordered in compartments and topologically associated domains5 and that the multiple levels of DNA folding generate contacts between different genomic regions that may involve chromatin loop formation6,7. The 3D dynamic looping of chromatin can influence many different biological processes such as transcription8,9, differentiation and development10,11, DNA repair12,13, while its perturbations are involved in various diseases14,15,16 and developmental defects15,17,18.
Many approaches have been developed to study the 3D genome organization. Chromosome conformation capture-based technologies (C-technologies, 3C, 4C, 5C, Hi-C and derivatives) have been developed to study genome organization in fixed cells3,4,19,20. Such approaches are based on the ability to capture the contact frequencies between genomic loci in physical proximity. C-technologies, depending on their complexity, catch the global 3D genome organization and nuclear topology of a cell population3,4,19,20. Nevertheless, 3D interactions are dynamic in time and space, highly variable between individual cells consisting of multiplex interactions, and are extensively heterogenous21,22.
3D multicolor DNA fluorescence in situ hybridization (FISH) is a technique that allows the visualization of specific genomic loci at a single cell level, enabling direct investigation of the 3D nuclear architecture in a complementary manner to C-technologies. It represents a technology currently used to unambiguously validate C-results. 3D multicolor DNA FISH uses fluorescently labeled probes complementary to the genomic loci of interests. The use of different fluorophores and suitable microscopy equipment allow contemporary visualization of multiple targets within the nuclear space23,24. In recent years, FISH has been combined with technological advances in microscopy to obtain the visualization of fine-scale structures at high resolution25,26 or with CRISPR-Cas approaches for the visualization of the nucleic acids in live imaging27,28. Despite wide adoption, the 3D multicolor DNA FISH approach is still considered difficult in many laboratories because the biological material used must be adapted.
Here, we provide a comprehensive protocol for 3D multicolor DNA FISH (from cell/probe preparation to data analysis) applicable to a wide range of human primary cells, enabling the visualization of multiple genomic loci and preserving the 3D structure of nuclei. In order to study nuclear architecture, the 3D structure of nuclei must be preserved. For this reason, contrasting from other existing protocols29,30,31, we avoid the use of an alcohol gradient and the storage of the coverslips in alcohol that can affect chromatin structure32. The method is adapted from preserved 3D DNA FISH protocols24,33 to be applied to a wide range of human primary cells, both isolated ex vivo or cultured in vitro. There are permeabilization and deproteinization parameters for different nuclear morphology and cytological characteristics (e.g., different degrees of nuclear compaction, cytoskeleton abundance)34. These parameters are often generally described in other protocols24,33, without providing a clear discrimination of the procedure within different cell types. Furthermore, we developed a specific tool named NuCL&#949;D (nuclear contacts locator in 3D)16, providing principles for data analysis that will improve the 3D proximity between different loci and their nuclear topological distribution within the nuclear space in an automated way.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>142475</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>140675</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Digoxigenin 488</td>
			<td>DBA</td>
			<td>DI7488</td>
			<td></td>
		</tr>
		<tr>
			<td>b-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>PeproTech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin 11 d-UTP</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0081</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA (bovine serum albumine)</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverlsips</td>
			<td>Marienfeld</td>
			<td>117500</td>
			<td></td>
		</tr>
		<tr>
			<td>CY3 d-UTP</td>
			<td>GE Healthcare</td>
			<td>PA53022</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4,6-diamidino-2-phenylindole)</td>
			<td>Thermo Fisher Scientific</td>
			<td>D21490</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonucleic acids single strand from salmon testes</td>
			<td>Sigma</td>
			<td>D7656</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran sulfate (powder)</td>
			<td>Santa Cruz</td>
			<td>sc-203917A</td>
			<td></td>
		</tr>
		<tr>
			<td>Digoxigenin 11 d-UTP</td>
			<td>Roche</td>
			<td>11093088910</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>21969-035 500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA polymerase I</td>
			<td>Thermo Fisher Scientific</td>
			<td>18010-017</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>AMPD1</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs (C-G-A-T)</td>
			<td>Euroclone</td>
			<td>BL0423A/C/G</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Sigma</td>
			<td>E9644.2MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma</td>
			<td>02860-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS Hyclone</td>
			<td>Thermo Fisher Scientific</td>
			<td>SH30109</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Sigma</td>
			<td>F8775-25mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Sigma</td>
			<td>F9037</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutammine</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030-024 100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G5516-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9510</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Sigma</td>
			<td>30721</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Cot-1 DNA</td>
			<td>Thermo Fisher Scientific</td>
			<td>15279-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Human</td>
			<td>Sigma</td>
			<td>I9278-5 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>63069</td>
			<td></td>
		</tr>
		<tr>
			<td>NaAc (Sodium Acetate, pH 5.2, 3 M)</td>
			<td>Sigma</td>
			<td>S2889</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (phosphate-buffered saline)</td>
			<td>Sigma</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Pennycillin/Streptavidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15070-063 100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepsin</td>
			<td>Biorad</td>
			<td>P6887</td>
			<td></td>
		</tr>
		<tr>
			<td>PhasePrep BAC DNA Kit</td>
			<td>Sigma</td>
			<td>NA0100-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine solution</td>
			<td>Sigma</td>
			<td>P8920</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Diamond Antifade Mountant</td>
			<td>Thermo Fisher Scientific</td>
			<td>P36970</td>
			<td></td>
		</tr>
		<tr>
			<td>PureLink Quick Gel Extraction &#38; PCR Purification Combo Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K220001</td>
			<td></td>
		</tr>
		<tr>
			<td>PureLink Quick Plasmid Miniprep Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K210010</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse cocktail</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM2288</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubbercement</td>
			<td>Bostik</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>VWR</td>
			<td>631-0114</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidina Alexa fluor 647</td>
			<td>Thermo Fisher Scientific</td>
			<td>S21374</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri-Sodium Citrate</td>
			<td>Sigma</td>
			<td>1110379026</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma</td>
			<td>T3253-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787-250mL</td>
			<td></td>
		</tr>
		<tr>
			<td>TWEEN 20</td>
			<td>Sigma</td>
			<td>P9416-100mL</td>
			<td></td>
		</tr>
	</tbody>
</table>

3d multicolor dna fish tool to study nuclear architecture in human primary cells

A major question in cell biology is genomic organization within the nuclear space and how chromatin architecture can influence processes such as gene expression, cell identity and differentiation. Many approaches developed to study the 3D architecture of the genome can be divided into two complementary categories: chromosome conformation capture based technologies (C-technologies) and imaging. While the former is based on capturing the chromosome conformation and proximal DNA interactions in a population of fixed cells, the latter, based on DNA fluorescence in situ hybridization (FISH) on 3D-preserved nuclei, allows contemporary visualization of multiple loci at a single cell level (multicolor), examining their interactions and distribution within the nucleus (3D multicolor DNA FISH). The technique of 3D multicolor DNA FISH has a limitation of visualizing only a few predetermined loci, not permitting a comprehensive analysis of the nuclear architecture. However, given the robustness of its results, 3D multicolor DNA FISH in combination with 3D-microscopy and image reconstruction is a possible method to validate C-technology based results and to unambiguously study the position and organization of specific loci at a single cell level. Here, we propose a step by step method of 3D multicolor DNA FISH suitable for a wide range of human primary cells and discuss all the practical actions, crucial steps, notions of 3D imaging and data analysis needed to obtained a successful and informative 3D multicolor DNA FISH within different biological contexts.

The method of 3D multicolor DNA FISH described in this article allows contemporary visualization of different genomic loci within preserved 3D nuclei (Figure 1B). This protocol permits the measurement of distances between alleles, and different genomic loci in order to evaluate their spatial proximity, and to assess their location within the nuclear space (e.g., loci distance from the centroid or the periphery of the nuclei)16. However, there are many...

Watch this Scientific Journal Video about 3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells at JoVE.com

3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells

3D multicolor DNA FISH represents a tool to visualize multiple genomic loci within 3D preserved nuclei, unambiguously defining their reciprocal interactions and localization within the nuclear space at a single cell level. Here, a step by step protocol is described for a wide spectrum of human primary cells.

A major question in cell biology is genomic organization within the nuclear space and how chromatin architecture can influence processes such as gene ...

Research

JoVE Journal

Genetics

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

ATAC-seq Assay with Low Mitochondrial DNA Contamination from Primary Human CD4+ T Lymphocytes

ATAC-seq has become a widely used methodology in the study of epigenetics due to its rapid and simple approach to mapping genome-wide accessible ...

Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and ...

Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis

Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis

Yersinia ruckeri is an important pathogen of farmed salmonids worldwide, but simple tools suitable for epizootiological investigations (infection tracing, ...

Navigating MARRVEL, a Web-Based Tool that Integrates Human Genomics and Model Organism Genetics Information

Through whole-exome/genome sequencing, human geneticists identify rare variants that segregate with disease phenotypes. To assess if a specific variant is ...

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported ...