We would like to thank Prof Michael Bittner for the kind gift of chromosome arm painting probes. LG was supported by EU funded EURO-Laminopathies project and the Brunel Progeria Research Fund.

<ol>
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H., Coverley, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+DNA+replication+and+the+nuclear+matrix.">Relationship between DNA replication and the nuclear matrix.</a> Genes Cells. 18 (1), 17-31 (2013).</li><li>Wilson, R. H. C., Coverley, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation-induced+changes+in+the+DNA-nuclear+matrix+interface,+revealed+by+high-throughput+analysis+of+DNA+halos.">Transformation-induced changes in the DNA-nuclear matrix interface, revealed by high-throughput analysis of DNA halos.</a> Science Reports. 7 (1), 6475 (2017).</li><li>Iarovaia, O. V., Akopov, S. B., Nikolaev, L. G., Sverdlov, E. D., Razin, S. 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U., 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=Farnesyltransferase+inhibitor+and+rapamycin+correct+aberrant+genome+organisation+and+decrease+DNA+damage+respectively,+in+Hutchinson-Gilford+progeria+syndrome+fibroblasts.">Farnesyltransferase inhibitor and rapamycin correct aberrant genome organisation and decrease DNA damage respectively, in Hutchinson-Gilford progeria syndrome fibroblasts.</a> Biogerontology. 19 (6), 579-602 (2018).</li><li>Telenius, 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=Degenerate+oligonucleotide-primed+PCR:+general+amplification+of+target+DNA+by+a+single+degenerate+primer.">Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer.</a> Genomics. 13 (3), 718-725 (1992).</li><li>Bridger, J. 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=Association+of+pKi-67+with+satellite+DNA+of+the+human+genome+in+early+G1+cells.">Association of pKi-67 with satellite DNA of the human genome in early G1 cells.</a> Chromosome Research. 6, 13-24 (1998).</li><li>Sales Gil, R., Vagnarelli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ki-67:+More+Hidden+behind+a+'Classic+Proliferation+Marker'.">Ki-67: More Hidden behind a 'Classic Proliferation Marker'.</a> Trends in Biochemical Sciences. 43 (10), 747-748 (2018).</li><li>Bikkul, M. U., 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=Telomere+elongation+through+hTERT+immortalization+leads+to+chromosome+repositioning+in+control+cells+and+genomic+instability+in+Hutchinson-Gilford+progeria+syndrome+fibroblasts,+expressing+a+novel+SUN1+isoform.">Telomere elongation through hTERT immortalization leads to chromosome repositioning in control cells and genomic instability in Hutchinson-Gilford progeria syndrome fibroblasts, expressing a novel SUN1 isoform.</a> Genes Chromosomes Cancer. 58 (6), 341-356 (2019).</li><li>Jackson, D. A., Cook, P. 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The authors have nothing to disclose.

The DNA halo method is an excellent method of choice when analyzing interactions between the nucleoskeleton and genome, however, there are some critical steps that must be adhered too. One of the most important parameters is the optimization of the cell seeding density. If cells become over confluent, then the DNA halos will overlap with neighboring cells making it impossible to perform the analysis. The CSK and extraction buffers must always be made fresh on the day of use with spermine, spermidine and digitonin being added to the extraction buffer at the end of the preparation process to maintain their biological activity. If performing Halo-FISH it is extremely important to use the correct denaturation temperature of the DNA halos to enable the probe or paint to subsequently hybridize.
Electron microscopy has been used to visualize the nuclear matrix, with filamentous structures being identified20. However, electron microscopy is limited as matrix associations with chromatin cannot easily be deduced. Indeed, the DNA Halo method is more versatile compared with electron microscopy as specific genes, chromosomes and cell states can all be examined. Furthermore, proteomic analysis of nuclear matrix proteins is being studied21,22. This method is good for comparing nuclear matrix components, particularly when comparing diseased cells, however, it doesn&#39;t provide the spatial distribution and attachments highlighted by the standard DNA Halo technique.
DNA Halo assays do have limitations. Firstly, as the matrix is extracted, this can only be performed on fixed cells so live imaging is not possible. Although the DNA Halo method is relatively quick and easy to perform, the overall process may be time consuming when cell culture, probe generation, Halo-FISH and analysis is all taken into account.
Image capture of DNA Halos and HALO-FISH using super-resolution microscopy would greatly improve the resolution of DNA specific probes and antibodies. In addition, as fluorochromes can be more easily spectrally resolved, it may be possible to use a number of DNA probes in a single experiment, providing even more information. Improvements in molecular biology techniques such as chromosome conformation capture (3C) have been used to determine interactions of gene loci and analyze the spatial organization on chromatin in the cell. DNA Halo assays and 3C can be combined, a term known as M3C23, again demonstrating the adaptability of the DNA Halo technique.
The original data presented here are to demonstrate the possibilities for genome behavior interrogation and how to present those data. With these data we have demonstrated that it is possible to determine significant differences in genome attachment using (1) chromosome painting probes, in this study revealing chromosome 18 being the least attached chromosome out of those analysed (Figure 3); (2) Gene loci with significant differences between two gene loci and (Figure 4) (3) Telomeres, which are less strongly attached in quiescent cells compared to proliferating and senescent cells (Figure 5). We are able to differentiate between proliferating and non-proliferating cells via the presence of the proliferation marker Ki67 antigen which is an insoluble protein so remains with the residual nuclei or using the incorporation of nucleotides to highlight cells that have been through S-phase within a specific time period (Figure 2). This technique has also enabled us to analyze genome behavior in cells that are compromised in their nucleoskeletons i.e. laminopathy cells and here and in Bikkul et al., 2018 we reveal that the genome can be less tightly attached when compared to control cells and can be restored when treating with specific drugs that ameliorate the effect of the lamin A mutation in classical HGPS cells15. However, we show new data here for the atypical HGPS AGO8466 cells, lacking a lamin A mutation but containing an unusual form of the LINC complex protein SUN119 that chromosome 13 is less tightly attached in (Figure 6).
HALO-FISH is a unique method by enabling the study of genomic interactions with the nucleoskeleton in combination with indirect immunofluorescence to resolve proteins not removed from the extraction procedure. It has been demonstrated that the nucleoskeleton is modified in various diseases such as certain cancer types19 and the importance of some nucleoskeleton-associated proteins as diagnostic biomarkers24,25. Thus, this technique has an important role in examining the effect of the nucleoskeleton on chromatin organization/disorganization in disease15,24,25,27 and is not restricted to human cells, with chromosomal painting probes from other animals, the same DNA-halo protocol could be employed28.

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/62017">Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci</a>. The Authors section was updated from:
Lauren S. Godwin1 
Joanna M. Bridger1 
Helen A. Foster2 
1Laboratory of Nuclear and Genomic Health, Centre for Genome Engineering and Maintenance, Division of Biosciences, Department of Life Sciences, College of Health, Medicine and Life Sciences, Brunel University London 
2Biosciences, Department of Clinical, Pharmaceutical and Biological Science, School of Life and Medical Sciences, University of Hertfordshire
to:
Lauren S. Godwin1 
Emily Roberts2 
Joanna M. Bridger1 
Helen A. Foster2 
1Laboratory of Nuclear and Genomic Health, Centre for Genome Engineering and Maintenance, Division of Biosciences, Department of Life Sciences, College of Health, Medicine and Life Sciences, Brunel University London 
2Biosciences, Department of Clinical, Pharmaceutical and Biological Science, School of Life and Medical Sciences, University of Hertfordshire

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/62017">Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci</a>. The Authors section was updated.

Erratum: Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci

The &#34;nuclear&#160;matrix&#34; was first described by Berezney and Coffey in 19741. After performing extractions with high salt molarities and nuclease treatment on rat liver nuclei, they identified a proteinaceous structural framework. The DNA halo procedure was subsequently adapted from this method and involves the removal of soluble proteins so that only the nuclear matrix (NM) and NM-associated proteins and chromosomes persist. DNA attachment regions are located at the base of DNA loops and are called matrix attached regions (MARs) or scaffold attachment regions (SARs), which are resistant to extraction with high salt concentrations and ionic detergent lithium-3,5-diiodosalicylate (LIS) respectively. In DNA halos, DNA associated with MARs/SARs are bound within the residual nucleus whereas the DNA loops extend away from these sites and form the DNA halo. We know now that the genome is anchored via lamina associated domains (LADs) to the nuclear lamina and through nucleolar associated regions (NADs) and possibly through other nuclear structures such as specific nuclear bodies.
The DNA halo method can be used for physical mapping of DNA, genes, and chromosomal regions as the extended DNA and chromatin provides a greater resolution because the chromatin is stripped of histones and the DNA is stretched out2,3,4,5,6. However, there are some limitations when using DNA halos for this application. For instance, DNA tightly associated with residual nuclei of DNA halos can be inaccessible to probes thus precluding it from analysis and physical mapping6. Other techniques such as fiber-FISH2,4,5,7 and molecular combing8 also enable physical mapping and have the advantage of being relatively quick and easy to perform. Both are preferentially used for DNA mapping of genes over DNA halos. These methods extract chromatin fibers via the use of solvent or salt extractions from the nucleus, however, molecular combing tends to have better reproducibility8,9.
There is an increasing evidence that the nucleoskeleton has a role in supporting key nuclear processes, such as attachment sites for DNA, chromatin remodeling, DNA transcription, DNA repair and DNA replication11,12. As such, the DNA halo technique was developed to investigate the interactions between the nucleoskeleton and genome during these cellular activities and has been routinely used and reported in research. This technique has also been used to investigate interactions between the genome and nucleoskeleton in relation to disease progression with malignancy-associated changes in nuclear structure being identified11.
The DNA halo technique has also been used to investigate the relationship between the genome and nucleoskeleton during development and differentiation12. A number of studies have used a variation of the DNA halo technique known as halosperm13 or SpermHalo-FISH if coupled with FISH14. Spermatozoa chromatin is tightly bound to proteins known as protamines and this technique was developed to improve access to the sperm DNA. Halosperm has been used to investigate the integrity of spermatozoa DNA and determine if DNA damage is present. Spermatozoa with less DNA damage correlate to a larger DNA halo size, whereas spermatozoa with increased levels of fragmented and damaged DNA had either small halos or none at all. Thus, halosperm can be used as a potential prognostic marker of embryo quality and successful pregnancy with IVF13. This example emphasizes the potential clinical applications of this technique. In our work we have used HALO-FISH to assess changes in genome behavior and the effect of specific drug treatments in the premature ageing disease Hutchinson-Gilford Progeria Syndrome (HGPS)15.
Together these, and other studies, highlight the breadth of processes/applications that the DNA halo technique can be used to study and utility of the technique.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10X PBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>10388739</td>
			<td>Used to create DNA halos</td>
		</tr>
		<tr>
			<td>5-bromo-2&#8242;-deoxy-uridine&#160;&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>B5002-100MG</td>
			<td>Labelled nucleotide</td>
		</tr>
		<tr>
			<td>5-Fluoro-2&#8242;-deoxyuridine</td>
			<td>Sigma-Aldrich</td>
			<td>F0503-100MG</td>
			<td>Labelled nucleotide</td>
		</tr>
		<tr>
			<td>Agar Technical</td>
			<td>Thermo Fisher Scientific</td>
			<td>15562141</td>
			<td>DNA isolation of BAC clones</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A939-50G</td>
			<td>Check product size of DOP-PCR and nick translation</td>
		</tr>
		<tr>
			<td>Atypical type 2 HGPS fibroblasts (AG08466)</td>
			<td>Coriell Institute</td>
			<td>AG08466</td>
			<td>Cell line</td>
		</tr>
		<tr>
			<td>Bacto tryptone</td>
			<td>Thermo Fisher Scientific</td>
			<td>16269751</td>
			<td>DNA isolation of BAC clones</td>
		</tr>
		<tr>
			<td>Biotin-16-dUTP</td>
			<td>Roche Diagnostics&#160;</td>
			<td>11093711103</td>
			<td>Labelled nucleotides</td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Sigma-Aldrich</td>
			<td>C0378-25G</td>
			<td>DNA isolation of BAC clones</td>
		</tr>
		<tr>
			<td>Classical Hutchinson-Gilford progeria syndrome (HGPS) fibroblasts (AG06297)&#160;</td>
			<td>Coriell Institute</td>
			<td>AG0297</td>
			<td>Cell line</td>
		</tr>
		<tr>
			<td>Coplin jar</td>
			<td>Thermo Fisher Scientific</td>
			<td>12608596</td>
			<td>Holds 5 slides or 8 slides back to back</td>
		</tr>
		<tr>
			<td>Cot-1 DNA</td>
			<td>Thermo Fisher Scientific</td>
			<td>15279011</td>
			<td>Block nonspecific hybridization in HALO FISH</td>
		</tr>
		<tr>
			<td>DEPC-treated water</td>
			<td>Sigma-Aldrich</td>
			<td>693520-1L</td>
			<td>DNA isolation of BAC clones</td>
		</tr>
		<tr>
			<td>Dextran sulphate</td>
			<td>Sigma-Aldrich</td>
			<td>S4030</td>
			<td>Hybridisation mixture</td>
		</tr>
		<tr>
			<td>Digitonin</td>
			<td>Sigma-Aldrich</td>
			<td>D141</td>
			<td>Component of extraction buffer</td>
		</tr>
		<tr>
			<td>Digoxigenin-11-dUTP&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>11093088910</td>
			<td>Labelled nucleotides</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse Cy3</td>
			<td>Jackson Laboratory</td>
			<td>715-165-150</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E6758</td>
			<td>Component of extraction buffer</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td>Component of extraction buffer</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>443611</td>
			<td>Probe precipitation and HALO FISH</td>
		</tr>
		<tr>
			<td>Fetal bovine system</td>
			<td>Thermo Fisher Scientific</td>
			<td>26140079</td>
			<td>Cell culture serum</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Thermo Fisher Scientific</td>
			<td>10523525</td>
			<td>2D FISH of DNA halos</td>
		</tr>
		<tr>
			<td>Glass wool</td>
			<td>Sigma-Aldrich</td>
			<td>18421</td>
			<td>Spin column</td>
		</tr>
		<tr>
			<td>Herring sperm</td>
			<td>Sigma-Aldrich</td>
			<td>D7290</td>
			<td>Probe precipitation</td>
		</tr>
		<tr>
			<td>HXP&#8482; Lamp (metal halide microscope lamp)</td>
			<td>OSRAM</td>
			<td>HXP-R120W45C VIS</td>
			<td>Image capture of DNA halos</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Thermo Fisher Scientific</td>
			<td>10313680</td>
			<td>Cleaning microscope slides</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma-Aldrich</td>
			<td>I9516-25ML</td>
			<td>DNA isolation of BAC clones</td>
		</tr>
		<tr>
			<td>KAPA HiFi PCR Kit</td>
			<td>KAPA Biosystems</td>
			<td>KK2103</td>
			<td>PCR Kit</td>
		</tr>
		<tr>
			<td>Leica DM4000 fluorescent microscope with DFC365 FX camera and LAS AF (Version: 4.5.0) image acquisition software.</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>Image capture of DNA halos</td>
		</tr>
		<tr>
			<td>Luria-Bertani agar&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>13274843</td>
			<td>DNA isolation of BAC clones</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td>Component of CSK buffer</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Thermo Fisher Scientific</td>
			<td>10284580</td>
			<td>Cleaning and sterilizing microscope slides</td>
		</tr>
		<tr>
			<td>Mouse anti-BrdU antibody</td>
			<td>BD Pharmingen</td>
			<td>B2531-100UL</td>
			<td>BrdU visualisation</td>
		</tr>
		<tr>
			<td>Newborn calf serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>16010159</td>
			<td>Cell culture serum and blocking reagent</td>
		</tr>
		<tr>
			<td>Nick translation kit</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PCR grade water</td>
			<td>Sigma-Aldrich</td>
			<td>693520-1L</td>
			<td>PCR and DNA isolation of BAC clones</td>
		</tr>
		<tr>
			<td>PCR Primers</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>Sigma-Aldrich</td>
			<td>P1851</td>
			<td>Component of CSK and extraction buffers</td>
		</tr>
		<tr>
			<td>Potassium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>P1190-100G</td>
			<td>DNA isolation of BAC clones</td>
		</tr>
		<tr>
			<td>QuadriPERM&#174; 4 X 12</td>
			<td>SARSTEDT</td>
			<td>94.6077.307</td>
			<td>Square cell culture dish, polysterene with four compartments.&#160; This has hydrophobic surface, is sterile, non-pyrogenic/endotoxin-fee and non-cytotoxic.</td>
		</tr>
		<tr>
			<td>Rabbit Anti-Ki67 antibody</td>
			<td>Sigma-Aldrich</td>
			<td>ZRB1007-25UL</td>
			<td>Proliferation marker</td>
		</tr>
		<tr>
			<td>Rnase A</td>
			<td>Sigma-Aldrich</td>
			<td>R6513</td>
			<td>DNA isolation of BAC clones</td>
		</tr>
		<tr>
			<td>Rubber cement</td>
			<td>Halford&#39;s</td>
			<td>101836</td>
			<td>2D FISH of DNA halos</td>
		</tr>
		<tr>
			<td>Sephadex G-50</td>
			<td>Sigma-Aldrich</td>
			<td>S6022-25G</td>
			<td>Spin column</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>S2889</td>
			<td>Probe precipitation</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td>Component of CSK, extraction and SSC buffers</td>
		</tr>
		<tr>
			<td>Sodium citrate</td>
			<td>Sigma-Aldrich</td>
			<td>C8532</td>
			<td>Component of SSC buffer</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulphate&#160;</td>
			<td></td>
			<td>L3771-100G</td>
			<td>DNA isolation of BAC clones</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>S8045-500G</td>
			<td>DNA isolation of BAC clones</td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>Sigma-Aldrich</td>
			<td>S2626</td>
			<td>Component of extraction buffer</td>
		</tr>
		<tr>
			<td>Spermine</td>
			<td>Sigma-Aldrich</td>
			<td>S4264</td>
			<td>Component of extraction buffer</td>
		</tr>
		<tr>
			<td>Streptavidin-Cy3&#160;</td>
			<td>Amersham Life Sciences Ltd, Scientific Laboratory Supplies</td>
			<td>pa43001</td>
			<td>Probe antibody</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td>Component of CSK buffer</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td>CSK buffer+A66:D68</td>
		</tr>
		<tr>
			<td>SuperFrost&#8482; microscope slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>12372098</td>
			<td>Microscope slides: 1 mm thickness, 76 mm length, 26 mm width. Uncoated.</td>
		</tr>
		<tr>
			<td>Swine anti-rabbit TRITC&#160;</td>
			<td>Dako</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TELO-PNA FISH KIT</td>
			<td>Agilent Dako</td>
			<td>K532511-8</td>
			<td>Delineation of telomeres</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma-Aldrich</td>
			<td>T3253-100G</td>
			<td>Column buffer</td>
		</tr>
		<tr>
			<td>Triton&#8482; X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td>Component of CSK buffer</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Thermo Fisher Scientific</td>
			<td>10158962</td>
			<td>DNA isolation of BAC clones</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416- 100ML</td>
			<td>Detergent</td>
		</tr>
		<tr>
			<td>Vectashield mountant containing DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td>2D FISH of DNA halos</td>
		</tr>
		<tr>
			<td>Whole human chromosome probes</td>
			<td>Calbiochem</td>
			<td></td>
			<td>2D FISH of DNA halos</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Thermo Fisher Scientific</td>
			<td>10108202</td>
			<td>DNA isolation of BAC clones</td>
		</tr>
	</tbody>
</table>

fluorescence in situ hybridization on dna halo preparations to reveal whole chromosomes, telomeres and gene loci

The genome is associated with several structures inside cell nuclei, in order to regulate its activity and anchor it in specific locations. These structures are collectively known as the nucleoskeleton and include the nuclear lamina, the nucleoli, and nuclear bodies. Although many variants of fluorescence in situ hybridization (FISH) exist to study the genome and its organization, these are often limited by resolution and provide insufficient information on the genome&#39;s association with nuclear structures. The DNA halo method uses high salt concentrations and nonionic detergents to generate DNA loops that remain anchored to structures within nuclei through attachment regions within the genome. Here, soluble nuclear proteins, such as histones, lipids, and DNA not tightly bound to the nuclear matrix, are extracted. This leads to the formation of a halo of unattached DNA surrounding a residual nucleus which itself contains DNA closely associated with internal nuclear structures and extraction-resistant proteins. These extended DNA strands enable increased resolution and can facilitate physical mapping. In combination with FISH, this method has the added advantage of studying genomic interactions with all the structures that the genome is anchored by. This technique, termed HALO-FISH, is highly versatile whereby DNA halos can be coupled with nucleic acid probes to reveal gene loci, whole chromosomes, alpha satellite, telomeres and even RNA. This technique provides an insight into nuclear organization and function in normal&#160;cells and in disease progression such as with cancer.

This method of DNA halo preparation has helped us in our endeavors to determine differences in genome behavior within young and old cells, but also in cells derived from premature ageing diseases with aberrant nucleoskeletal proteins15. Figure 1 displays examples of DNA halos where it is possible to see the edge of a residual nucleus, the DNA remaining within the residual nucleus and the unattached DNA that has spooled out into the surrounding area creating a DNA halo. It also depicts the analysis showing how the residual nucleus is obtained and the NE and CTE measurements. It is possible to differentiate between proliferating and non-proliferating cells by either incorporating a labeled nucleotide such as BrdU when cells are in S-phase or employing the diagnostic proliferation marker anti-pKi67, which reveals nucleoli, and regions of heterochromatin in G1 cells17,18. Primary cells grown in high serum without achieving confluency, that are negative for the proliferation markers, are assumed to be senescent. Primary cells grown in low serum or have become confluent i.e., contact inhibited that are negative for the proliferation markers are deemed quiescent and would be able to reenter the proliferative cell cycle given the correct nutrients and situation. Being able to differentiate between Ki67 positive and negative cells has enabled us to determine differences between proliferating, quiescent and senescent human dermal fibroblasts. Figure 2 displays DNA halos of proliferating human dermal fibroblasts created from cells where BrdU was incorporated into them during DNA replication, a mechanism that does not occur in non-proliferating cells, and subsequently stained with anti-BrdU antibody. Staining with the proliferative marker anti-pKi67 antibody is also visible in Figure 2. This is a robust antigen and survives the FISH protocol and so can be stained for post-FISH and pre-mounting. Thus, proliferating cells are positive (red) for BrdU and anti-pKi67 (red) in the left-hand column and non-proliferating cells, indeed senescent cells in Figure 2 are displayed in the right-hand column. The green signals are individual telomeres revealed with a telomere PNA FISH/FITC kit. Combining immunofluorescence with DNA halos enables analysis during different cell states, as shown in Figure 2 when investigating proliferating, quiescent and senescent cells. Depending on the antibody chosen other conditions can be examined, such as differentiation, DNA damage via irradiation etc.
Chromosome territories can also be visualized within DNA halos using FISH. Due to the preparation permitting spooling of DNA out the nuclei, the chromosome territory shape can be disturbed, with smaller or larger amounts of the chromosome found in the DNA halo, depending on the anchorage of the genome inside the residual nucleus and its structures. Figure 3 reveals a panel of DNA halos whereby individual chromosomes have been revealed with specific whole arm chromosome painting probes (red) for chromosomes 1, 13, 17 and 18. Anti-pKi67 (green) has been used to mark proliferating cells and its absence within the same culture, upon the same slide, denoting senescent cells. It is very obvious from the images and the data presented as CTE/NE that the small gene-poor chromosome 18 is a chromosome that has few attachments and spools further out into the DNA halo away from the residual nuclei and is significantly further from the center of the residual nuclei than the other chromosomes. However, this is also true for chromosome 1 as well. Using the proliferative marker anti-pKi67 it has also been possible to compare proliferating with senescent cells, within the same culture, and on the same slide, and this analysis has revealed that chromosomes within these two very different cell statuses are not significantly different from one another, with respect to attachment with the residual nuclear structures.
Interestingly, genes also are showing statistically significant differences between proliferating and senescent cells with respect to remaining within a residual nucleus or being located in the DNA Halo. Figure 4 demonstrates this with gene loci delineated by labeled BAC probes in red and anti-Ki67 in green. There are no significant differences between gene locations in the proliferating versus the senescent cells, after a DNA Halo preparation. However, there are significantly more catenin alpha 1 CTNNA1 loci within the DNA halo than cyclin D1 CNDD1 loci, where there are very few. Figure 5 displays DNA halo preparations with telomeres in green. The background is left deliberately high to enable telomere signals to be visualized within the DNA halo. In this set of data quiescent cells i.e., cells that have been serum starved for 7 days have been included and interestingly there are significantly more telomeres unattached and located within the DNA halos in quiescent cells than for proliferating and senescent cells. In Figure 5a the proportion of telomeres in the DNA halo can be observed, particularly for the image &#39;Experiment 2&#39;. This corresponds with Figure 5b where the mean percentage of telomeres in DNA halo is approximately 17% in quiescent cells. There is some evidence that not all telomeres in senescent cells can be seen as some of them maybe very short.
This method of DNA halo has been successful for us to investigate genome interaction alterations within nuclei in diseased cells15. Figure 6 demonstrates differences in chromosome attachment in primary control fibroblasts and in diseased cells with typical (lamin A mutation) and atypical Hutchinson-Gilford Progeria Syndrome, expressing a different SUN1 isoform and no lamin A mutation19. Chromosomes 1 and 13 show statistically significant differences in their attachment within the residual nuclei when compared to control DNA halos. Figure 6b correlates the position of the whole chromosome territory to the residual nucleus and DNA Halo. Values of 1 or less indicates the chromosome is located within the residual nucleus and values over 1 demonstrate chromosomes or portions of chromosomes within the DNA Halo.
Overall, this highlights the utility of HALO-FISH in investigating genomic interactions of whole chromosomes, specific genes and telomeres under a variety of conditions that affect the cell cycle (proliferation, quiescence and senescence) or within disease cells e.g., progeria and cancer cell lines. Indeed, the differences in interactions between these states implies the nucleoskeleton has an important role in regulating key processes within the nucleus.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62017/62017fig01.jpg" /> 
Figure 1:&#160;HDF extracted nucleus displaying the residual nucleus and DNA halo and overview of analysis method. (a) An HDF nucleus prepared via DNA halo assay and counterstained with DAPI. The brightly stained residual nucleus shows DNA anchored to the nucleoskeleton and this is surrounded by the non-attached DNA which forms a halo of DNA. Magnification = x 100; scale bar 10 &#181;m. (b) The blue channel captures the DAPI-stained nucleus and surrounding DNA. The residual nucleus is selected and removed using ImageJ. The arrow depicts the distance from the nuclear center to the residual nuclear edge (NE). (c) The red channel shows the probe signal. (d) The image denoted &#39;Result&#39; is the outcome of superimposing the red channel on the blue channel image; this allows the distance from the nuclear center to the furthest chromosome territory edge (CTE). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62017/62017fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62017/62017fig02.jpg" /> 
Figure 2:&#160;DNA halo preparation with telomere PNA FISH on proliferating and senescent HDFs. Telomere PNA FISH on HDFs subjected to DNA halo assay. Telomere signals are visualized in green (FITC), residual and halo DNA was counterstained using DAPI (blue) and proliferating nuclei were detected using either anti-BrdU or anti-pKi67 antibodies via indirect immunofluorescence in red (TRITC). Magnification = x 100; scale bar 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62017/62017fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62017/62017fig03.jpg" /> 
Figure 3:&#160;Nucleoskeleton-chromosome interactions and analysis using DNA halo assay. (a) 2D-FISH with probes specific for chromosomes 1, 13, 15, 17 and 18 was performed on HDFs subjected to DNA halo preparation. Whole chromosomes were painted in red (Cy3) and nuclei were probed with pKi67 to determine if they were proliferating or senescent. Proliferating cells (pKi67+) were delineated in green (FITC), whereas senescent cells remained unstained (pKi67-) i.e. no green signal detected. Magnification = x 100; scale bar 10 &#181;m. (b) Chromosome anchorage by the nucleoskeleton in proliferating and senescent HDFs that had undergone HALO-FISH. Measurements show the ratio of the furthest chromosome territory edge (CTE) to respective nuclear edge (NE) for chromosomes 1, 13, 15, 17 and 18 in proliferating (pKi67+) and senescent (pKi67-) cells. Error bars represent &#177; SEM. (c) Modified box plot representation of chromosome territory edge (CTE) to respective nuclear edge (NE) of specific chromosomes in pKi67+ and pKi67- nuclei. Q1 = lower quartile; Min = lowest value recorded; Med = median; Max = maximum value recorded; Q3 = upper quartile. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62017/62017fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62017/62017fig04.jpg" /> 
Figure 4:&#160;Gene-specific interactions in HDFs using HALO-FISH. (a) DNA halo extracted nuclei were probed with gene specific probes (CCND1 and CTNNA1) to investigate their anchorage to the NM on proliferating and senescent cells. The gene signals are shown in red (Cy3) and anti-pKi67 depicts proliferating cells and signal is visualized in green (FITC). For the proliferating CCND1 image, the residual nucleus is enclosed within the white circle, and the space between the white and green circle depicts the DNA Halo. Magnification = x 100; scale bar 10 &#181;m. (b) Gene-specific signals for CCND1 and CTNNA1 are compared between the residual nucleus and DNA halo, and also, between proliferating and senescent cells. Error bars represent &#177; SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62017/62017fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62017/62017fig05.jpg" /> 
Figure 5:&#160;DNA halo assay on quiescent HDFs probed with telomere PNA-FISH. (a) Quiescence of HDFs was induced by culture in low serum medium for 7 days. The DNA halo assay was performed, and PNA-FISH enabled visualization of telomeres by FITC signal (green) and the residual nucleus and surrounding DNA halo was counterstained with DAPI (blue). Cells were also stained with anti-pKi67 antibody to ensure nuclei were non-proliferating. This was repeated on two separate occasions. Magnification = x 100; scale bar 10 &#181;m. (b) Comparison of the mean percentage of telomeres localized within the DNA halo in proliferating, senescent and quiescent HDF cells. Error bars represent &#177; SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62017/62017fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62017/62017fig06.jpg" /> 
Figure 6: Examining whole chromosome anchorage to the nucleoskeleton in HGPS cells using HALO-FISH26. (a) Control HDF (2DD), classical HGPS (AG06297) and atypical type 2 HGPS (AG08466) nuclei underwent DNA halo preparation and then 2D-FISH using whole chromosome paints for chromosome 1, 13, 15 and 17. Whole chromosomes are depicted in green (FITC) and DNA was counterstained with DAPI (blue). Magnification = x 100; scale bar 10 &#181;m. (b) Positioning of chromosomes within extracted nuclei was determined by measuring the ratio of the mean chromosome territory edge (CTE) to the nuclear edge (NE). A ratio above 1 demonstrates that the furthest CTE lies outside the corresponding NE within the DNA halo, while a ratio below 1 signifies that the furthest CTE lies within the NE within the residual nucleus. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62017/62017fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Constituents</td>
			<td>Volume(&#956;L)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5XDOP-PCRbuffer</td>
			<td>10</td>
		</tr>
		<tr>
			<td>dNTPmix(withoutdTTP)(2mM)</td>
			<td>5</td>
		</tr>
		<tr>
			<td>dTTP(2mM)</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Biotin-16-dUTPorDigoxigenin-11-dUTP</td>
			<td>10</td>
		</tr>
		<tr>
			<td>DOPprimer(20&#956;M)</td>
			<td>5</td>
		</tr>
		<tr>
			<td>TaqDNAPolymerase(1U/&#956;L)</td>
			<td>1</td>
		</tr>
		<tr>
			<td>PCRgradewater</td>
			<td>12</td>
		</tr>
		<tr>
			<td>Template</td>
			<td>5</td>
		</tr>
	</tbody>
</table>
Table 1: Table showing the DOP-PCR components and volumes for a 1x reaction
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step&#160;</td>
			<td>Cycles&#160;</td>
			<td>Temp (degree Centigrade)</td>
			<td>Time&#160;</td>
		</tr>
		<tr>
			<td>Initial Denaturation&#160;</td>
			<td>1</td>
			<td>95</td>
			<td>3 min</td>
		</tr>
		<tr>
			<td>Denaturation&#160;</td>
			<td rowspan="3">34</td>
			<td>98</td>
			<td>20 s</td>
		</tr>
		<tr>
			<td>Primer Annealing&#160;</td>
			<td>62</td>
			<td>1 min</td>
		</tr>
		<tr>
			<td>Extension&#160;</td>
			<td>72</td>
			<td>30 s</td>
		</tr>
		<tr>
			<td>Final Extension&#160;</td>
			<td>1</td>
			<td>72</td>
			<td>5 min&#160;</td>
		</tr>
		<tr>
			<td>Cooling&#160;</td>
			<td></td>
			<td>4</td>
			<td>Hold</td>
		</tr>
	</tbody>
</table>
Table 2: Table showing the DOP-PCR cycle, temperature, and time profile.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Constituent</td>
			<td>Volume(&#956;L)</td>
		</tr>
		<tr>
			<td>10x NT buffer (0.5M Tris-HCl pH 8,50 mM MgCl2, 0.5 mg/ml BSA)</td>
			<td>5</td>
		</tr>
		<tr>
			<td>0.1 M beta-mercaptoethanol</td>
			<td>5</td>
		</tr>
		<tr>
			<td>10X Nucleotide stock (0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.5 mM dTTP, 0.5 mg/ml biotin-16-dUTP)</td>
			<td>5</td>
		</tr>
		<tr>
			<td>Dnase I (1 ng/ml)</td>
			<td>2</td>
		</tr>
		<tr>
			<td>DNApolymerase I</td>
			<td>5U per &#956;g of DNA</td>
		</tr>
		<tr>
			<td>DNAtemplate (1 &#956;g)</td>
			<td>1</td>
		</tr>
		<tr>
			<td>DEPC-treated water</td>
			<td>To 50 &#956;L</td>
		</tr>
	</tbody>
</table>
Table 3: Table showing the nick translation components and volumes for a one probe.

Watch this Scientific Journal Video about Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci at JoVE.com

Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci

Combining DNA halo preparations with fluorescence in situ hybridization enables high-resolution analysis of genomic interactions with the nucleoskeleton. Attached genome leads to hybridized fluorescent signals located within the residual extracted nuclei, whereas non-attached genome is in the halo of DNA surrounding the residual nuclei.

The genome is associated with several structures inside cell nuclei, in order to regulate its activity and anchor it in specific locations. These ...

fluorescence-situ-hybridization-on-dna-halo-preparations-to-reveal

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JoVE Journal

Biology

2.8K Views.  Brunel University London. Combining DNA halo preparations with fluorescence in situ hybridization enables high-resolution analysis of genomic interactions with the nucleoskeleton. Attached genome leads to hybridized fluorescent signals located within the residual extracted nuclei, whereas non-attached genome is in the halo of DNA surrounding the residual nuclei. 

Video: Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci

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Chapters in this video

Double Whole Mount in situ Hybridization of Early Chick Embryos

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos