Name: functional imaging of viral transcription factories using 3d fluorescence microscopy
Uploaded: 2018-01-18T14:00:00
Description: Watch this Scientific Journal Video about Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy at JoVE.com

This research was supported by National Institutes of Health grant (R01-DE025985) and by an American Cancer Society Research Scholar Award (RSG-13-383-MPC). This work was also supported by grants from the U.S. Department of Agriculture (2015-67015-23268 and 2014-67015-21787) and New Research Initiative Grant from University of California, Davis.

CAUTION: Cell lines used in this procedure contain infectious viruses, be careful and only proceed in level 2 BSL facility or higher.
1. Cell Preparation and Maintenance
<ol>
	<li>Culture TREx-K-RTA BCBL-1 cells (or another KSHV infected PEL cell lines) in RPMI1640 medium supplemented with 15% fetal bovine serum (FBS) and 1% penicillin streptomycin glutamine solution.</li>
	<li>Grow cells at 37 &#176;C with 5% carbon dioxide (CO2), making sure to continuously grow and to split cel...

<ol>
	<li>de Laat, W., Grosveld, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grosveld+Spatial+organization+of+gene+expression:+the+active+chromatin+hub.">Grosveld Spatial organization of gene expression: the active chromatin hub.</a> Chromosome Res. 11 (5), 447-459 (2003).</li><li>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=Protein+dynamics:+implications+for+nuclear+architecture+and+gene+expression.">Protein dynamics: implications for nuclear architecture and gene expression.</a> Science. 291 (5505), 843-847 (2001).</li><li>Bernardi, R., Pandolfi, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure,+dynamics+and+functions+of+promyelocytic+leukaemia+nuclear+bodies.">Structure, dynamics and functions of promyelocytic leukaemia nuclear bodies.</a> Nat Rev Mol Cell Biol. 8 (12), 1006-1016 (2007).</li><li>Cioce, M., Lamond, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cajal+bodies:+a+long+history+of+discovery.">Cajal bodies: a long history of discovery.</a> Annu Rev Cell Dev Biol. 21, 105-131 (2005).</li><li>Boisvert, F. M., van Koningsbruggen, S., Navascues, J., Lamond, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multifunctional+nucleolus.">The multifunctional nucleolus.</a> Nat Rev Mol Cell Biol. 8 (7), 574-585 (2007).</li><li>Sutherland, H., Bickmore, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factories:+gene+expression+in+unions.">Transcription factories: gene expression in unions.</a> Nat Rev Genet. 10 (7), 457-466 (2009).</li><li>Schmid, M., Speiseder, T., Dobner, T., Gonzalez, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+virus+replication+compartments.">DNA virus replication compartments.</a> J Virol. 88 (3), 1404-1420 (2014).</li><li>Chen, C. 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=Kaposi's+Sarcoma-Associated+Herpesvirus+Hijacks+RNA+Polymerase+II+To+Create+a+Viral+Transcriptional+Factory.">Kaposi's Sarcoma-Associated Herpesvirus Hijacks RNA Polymerase II To Create a Viral Transcriptional Factory.</a> J Virol. 91 (11), (2017).</li><li>Ganem, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KSHV+and+the+pathogenesis+of+Kaposi+sarcoma:+listening+to+human+biology+and+medicine.">KSHV and the pathogenesis of Kaposi sarcoma: listening to human biology and medicine.</a> J Clin Invest. 120 (4), 939-949 (2010).</li><li>Mesri, E. A., Cesarman, E., Boshoff, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kaposi's+sarcoma+and+its+associated+herpesvirus.">Kaposi's sarcoma and its associated herpesvirus.</a> Nat Rev Cancer. 10 (10), 707-719 (2010).</li><li>Campbell, 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=Protein+arginine+methyltransferase+1-directed+methylation+of+Kaposi+sarcoma-associated+herpesvirus+latency-associated+nuclear+antigen.">Protein arginine methyltransferase 1-directed methylation of Kaposi sarcoma-associated herpesvirus latency-associated nuclear antigen.</a> J Biol Chem. 287 (8), 5806-5818 (2012).</li><li>Guito, J., Lukac, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KSHV+Rta+Promoter+Specification+and+Viral+Reactivation.">KSHV Rta Promoter Specification and Viral Reactivation.</a> Front Microbiol. 3, 30 (2012).</li><li>Darst, R. P., Haecker, I., Pardo, C. E., Renne, R., Kladde, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+diversity+of+Kaposi's+sarcoma-associated+herpesvirus.">Epigenetic diversity of Kaposi's sarcoma-associated herpesvirus.</a> Nucleic Acids Res. 41 (5), 2993-3009 (2013).</li><li>Osborne, C. 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=Myc+dynamically+and+preferentially+relocates+to+a+transcription+factory+occupied+by+Igh.">Myc dynamically and preferentially relocates to a transcription factory occupied by Igh.</a> PLoS Biol. 5 (8), 192 (2007).</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>Borah, S., Darricarrere, N., Darnell, A., Myoung, J., Steitz, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+viral+nuclear+noncoding+RNA+binds+re-localized+poly(A)+binding+protein+and+is+required+for+late+KSHV+gene+expression.">A viral nuclear noncoding RNA binds re-localized poly(A) binding protein and is required for late KSHV gene expression.</a> PLoS Pathog. 7 (10), 1002300 (2011).</li><li>Chang, P. C., 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=Histone+demethylase+JMJD2A+regulates+Kaposi's+sarcoma-associated+herpesvirus+replication+and+is+targeted+by+a+viral+transcriptional+factor.">Histone demethylase JMJD2A regulates Kaposi's sarcoma-associated herpesvirus replication and is targeted by a viral transcriptional factor.</a> J Virol. 85 (7), 3283-3293 (2011).</li><li>McAllister, S. C., Hansen, S. G., Messaoudi, I., Nikolich-Zugich, J., Moses, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+efficiency+of+phorbol+ester-induced+lytic+reactivation+of+Kaposi's+sarcoma-associated+herpesvirus+during+S+phase.">Increased efficiency of phorbol ester-induced lytic reactivation of Kaposi's sarcoma-associated herpesvirus during S phase.</a> J Virol. 79 (4), 2626-2630 (2005).</li><li>Miller, G., El-Guindy, A., Countryman, J., Ye, J., Gradoville, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lytic+cycle+switches+of+oncogenic+human+gammaherpesviruses.">Lytic cycle switches of oncogenic human gammaherpesviruses.</a> Adv Cancer Res. 97, 81-109 (2007).</li><li>Guito, J., Lukac, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KSHV+Rta+Promoter+Specification+and+Viral+Reactivation.">KSHV Rta Promoter Specification and Viral Reactivation.</a> Front Microbiol. 3, 30 (2012).</li></ol>

There are some aspects of the protocol that can be altered to accommodate unusual circumstances. The choice of fixation and permeabilization buffers can also be altered. For fixation buffers, paraformaldehyde is also effective and ethanol can be used for permeabilization. 
Quenching the formaldehyde with glycine PBS is recommended as it prevents formaldehyde from disabling the antibodies in absence of bovine serum albumin (BSA), but if the coverslips are washed thoroughly enough, the glycine P...

It has become increasingly clear that the spatiotemporal organization of the nucleus plays an important role in modulating finely-tuned gene expression in eukaryotes. Most tissue specific genes are distributed over many chromosomes and need to be regulated synchronously in order to respond to specific stimuli in a coordinated manner1. Cells construct active chromatin hubs (ACH) as a way of bringing together genes and their cis-regulatory components to a specific nuclear space1.
It has also been demonstrated by various studies that although the nucleus seems very dense and viscous, biologically active molecules can traverse the nucleus rather quickly via diffusion2. As a consequence of this ephemeral property, most DNA binding proteins &#39;jump&#39; from binding site to binding site, feeling their way around the nuclear space, which allows for a highly adaptive and versatile nucleus2.
Despite this dynamic behavior of biomolecules within the nucleus, nuclear bodies without membranes such as (but not limited to) the nucleolus, Cajal bodies, and promyelocytic leukemia nuclear bodies (PML-NB) still exist. It is through a variety of mechanisms such as tandem DNA repeats (nucleolus), rRNA (nucleolus) and structural proteins such as coilin (Cajal bodies) or PML proteins (PML-NB) that hold these constructs together3,4,5. These structures and other congregations such as transcription factories serve as scaffolds that not only increase the local concentration of required components, but also regulate the composition of proteins and nucleic acids within them to ultimately create a central site for efficient cellular functions6.
Visualizing when and where nuclear structures form provides a wealth of information to researchers studying epigenetics. From a virology perspective, the reactivation of latently infected viruses, such as KSHV, significantly alters the landscape of the nucleus and distribution of nuclear enzymes to shift transcription primarily from cellular genes to viral genes, ultimately to produce fully functional viral progeny7,8. How does KSHV manipulate the cellular gene expression machinery to facilitate viral gene expression? Such information could also shed light on temporal cellular gene regulatory mechanisms.
Like all other herpesviruses, KSHV has two life cycles called lytic replication and latency. KSHV primarily resides in the latency stage, in which most of its viral gene expression is silenced, except latency associated genes9,10. During latency KSHV produces latency associated nuclear antigen (LANA), which constitutively binds the viral genomes and tethers viral chromatin to the human chromosome11. Because of LANA&#39;s intimate relationship with the viral genome, it is possible to use IFA and DAPI to stain and locate where the viral episomes were in relation to the host chromatin.
To study KSHV reactivation at the single episome level and the association with other viral episomes in an infected cell, a strategy to locate actively transcribing viral chromatin in situ has been established. Accordingly, LANA and RNAPII IFA with intron RNA-FISH were combined, by generating RNA-FISH probes that bind to the intron region (exact probe sequences can be found in the Izumiya lab&#39;s most recent publication8) of KSHV K-Rta-the key viral protein that is essential and sufficient for KSHV reactivation-it was possible to identify where transcription was actually taking place8,11,12,13,14,15. This intron RNA-FISH technique enables researchers to visualize where mRNA is being transcribed immediately before it is spliced and exported to cytoplasm16,17.
KSHV can be reactivated by various chemical stimuli including phorbol esters such as 12-O-Tetradeconoyl-phorbol-13acetate (TPA) and histone deacetylase inhibitors such as sodium butyrate, additionally KSHV can be induced to reactivate by overexpression of the viral transcription factor, K-Rta19. Researchers have successfully increased the efficiency of KSHV reactivation by synchronizing the cell&#39;s cycles prior to inducing reactivation18. Thus, for these particular studies, cells were synchronized using a double thymidine block (protocol described below) and incubated with TPA and doxycycline (Dox) for a short time. Doxycyline was used because the cell line utilized in these experiments has a doxycycline-inducible K-Rta cassette, which was cloned from cDNA and does not include the intron region of K-Rta. Although it is possible to reactivate KSHV using only induced K-Rta expression, it has been proven by other researchers that due to a variety of different biochemical factors K-Rta expression alone proves to be a feeble reactivation stimuli20. By combining all of these, and by limiting the drug incubations to a short period of time, a robust but not overly artificial KSHV reactivation was achieved for imaging.
After labeling LANA, RNAPII, K-Rta introns, and DNA as described in this paper, 3D fluorescence imaging was performed using a widefield deconvolution microscope. After processing with imaging software, the spatial distribution of active viral episomes can be properly evaluated. Using this technique, the central questions regarding the fundamental nature of the formation of active chromatin hubs and other nuclear structures can be studied. Having identical viral episomes in a single cell that processes the same regulatory elements may represent a unique research tool to deepen the understanding of spatiotemporal gene regulatory mechanisms.
A limitation of imaging multiple cell specimens fixed at different time points to characterize an inherently dynamic molecular process is that subtle or small-scale changes in fluorescence distribution are undetected or deemed insignificant. This is true unless in the rare instance, every cell observed displays the same subtle change. Thus, the full spatiotemporal relationship of active viral transcription and other nuclear structures cannot be critically evaluated using fixed imaging. To address these technical challenges, the best approach is to image live cells that have marked viral episomes and to follow the location of key cellular enzymes over time.

<table border="0" cellpadding="0" cellspacing="0" style="width:572px;" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">RPMI medium 1650 GlutaMAX</td>
			<td style="width:71px;">Gibco by Life Technologies</td>
			<td style="width:119px;">61870-036</td>
			<td style="width:167px;">Needs to be warmed to 37 &#176;C</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:71px;">Omega Scientific Inc.</td>
			<td style="width:119px;">FB-11</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Pen Strep Glutamine (100x)</td>
			<td style="width:71px;">Gibco by Life Technologies</td>
			<td style="width:119px;">10378-016</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Thymidine&#160;</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">T9250</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Dulbecco&#8217;s Phosphate Buffered Solution</td>
			<td style="width:71px;">Gibco by Life Technologies</td>
			<td style="width:119px;">14190-144</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">TPA</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">P1585</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Sodium Butyrate&#160;</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">B5887</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Formaldehyde Solution&#160;</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td align="right" style="width:119px;">252549</td>
			<td style="width:167px;">Corrosive, toxic, health hazard</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Diethyl Pyrocarbonate</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">D-5758</td>
			<td style="width:167px;">Acute toxicity&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Glycine</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td align="right" style="width:119px;">410225</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Acetone</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td align="right" style="width:119px;">179124</td>
			<td style="width:167px;">Flammable, toxic</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Methanol</td>
			<td style="width:71px;">Fisher Chemical&#160;</td>
			<td style="width:119px;">67-56-1</td>
			<td style="width:167px;">Flammable, toxic, health hazard</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Rat monoclonal antibody to KSHV/HHV-8 ORF73</td>
			<td style="width:71px;">Advanced Biotechnologies</td>
			<td style="width:119px;">13-210-100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-RNA Polymerase II Antibody clone CTD4H8</td>
			<td style="width:71px;">EMD Milipore</td>
			<td style="width:119px;">05-623</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Ribonucleic acid, transfer from baker&#8217;s yeast (S. cerevisiae)&#160;</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">9014-25-9</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Saline sodium citrate buffer 20x (SSC)</td>
			<td style="width:71px;">Thermo Fisher Scientific&#160;</td>
			<td align="right" style="width:119px;">15557044</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Formamide&#160;</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td align="right" style="width:119px;">295876</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Omnipur Dextran Sulfate, 50% solution</td>
			<td style="width:71px;">EMD Milipore</td>
			<td style="width:119px;">1916C093</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">DAPI (4&#8217;,6-diamindino-2-phenylindole, Dihydrochloride)</td>
			<td style="width:71px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:119px;">D1306</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">slowFade Gold antifade reagent</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:119px;">S36936</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Micro cover glass 22x22</td>
			<td style="width:71px;">Matsunami</td>
			<td style="width:119px;">C022221</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">VoloCITY</td>
			<td style="width:71px;"></td>
			<td style="width:119px;">3D imaging software</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">DeltaVision microscope</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Superfrost/Plus microscope slides</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">12-550-15</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

functional imaging of viral transcription factories using 3d fluorescence microscopy

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to understand where and when transcription is taking place within nuclear space and to visualize the relationship between episomes infected within the same cell's nucleus. Here, both immunofluorescence (IFA) and RNA-FISH have been combinedto identify actively transcribing Kaposi's sarcoma-associated herpesvirus (KSHV) episomes. By staining KSHV latency-associated nuclear antigen (LANA), it is possible to locate where viral episomes exist within the nucleus. In addition, by designing RNA-FISH probes to target the intron region of a viral gene, which is expressed only during productive infection, nascent RNA transcripts can be located. Using this combination of molecular probes, it is possible to visualize the assembly of large viral transcription factories and analyze the spatial regulation of viral gene expression during KSHV reactivation. By including anti-RNA polymerase II antibody staining, one can also visualize the association between RNA polymerase II (RNAPII) aggregation and KSHV transcription during reactivation.

A slightly abridged protocol was performed where BCBL-1 cells were used and only LANA and K-Rta RNA were stained (Figure 1). This experiment allows us to examine where actively transcribing viral episomes are and the heterogeneity of response to KSHV reactivation stimuli in a population of cells. In the cell indicated by the arrow in Figure 1, the distinct K-Rta fluorescence in regions that closely match the distribution of viral...

Watch this Scientific Journal Video about Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy at JoVE.com

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...

The overall goal of combining nascent RNA-FISH with amino staining of viral latent proteins is to visualize active transcription coupled to viral transcription factory formation. This method can be used to help answer key questions in the fields of genetics, cell biology, virology, and cell biology virology. The main advantage of this technique is it utilizes an inducible viral mini-chromosome model to study the molecular details behind genomic architectural alterations.Collect cells in a 1.5 milliliter micro-centrifuge tube. Centrifuge the cells for two minutes at 200 Volt gravity. Wash the cells three times with one milliliter of sterile DEPC PBS.Next, re-suspend the cells in 1.2 milliliters of DEPC PBS. Place the cover slips into the bottoms of a six-well plate. Pipette 200 microliters of the cell DEPC PBS mixture onto each cover slip.After the cells have settled for two minutes, aspirate excess DEPC PBS, leaving only a thin layer of cells. Carefully add one milliliter of fixing solution to each cover slip. Allow the cells to fix for 10 minutes.Wash the cover slips three times with DEPC PBS. After washing, carefully add one milliliter of glycine DEPC PBS to a final concentration of glycine of 100 millimolar to each cover slip. Allow the cells to quench for five minutes.Add 1.5 milliliters of DEPC PBS and place on a shaker for five minutes, and shake firmly but gently in hand for one minute. Aspirate the excess DEPC PBS and be careful not to disrupt the cells. Carefully add one milliliter of permeabilizing solution to each cover slip.Allow the cells to permeabilize for 15 minutes. Add 1.5 milliliters of DEPC PBS. Place the cells on a shaker for five minutes, and shake firmly but gently in hand for one minute.Aspirate the excess DEPC PBS and be careful not to disrupt the cells. Label the microscope slides, and moisten a paper towel then line the bottom of a sealable plastic container with the towel. Next, prepare the primary antibody solution and multiply the recipe for each cover slip present.Place 30 microliters of primary antibody solution onto each labeled microscope slide. Ensure the primary antibody concentration is correct by holding the corner of the cover slip and gently flicking it. Additionally, the corner of a tissue can be used to wick excess moisture.Then place the cover slip onto the labeled microscope slides, being careful not to introduce bubbles. Cover the exterior of the container with plastic wrap. Move the container into an incubator set to 37 degrees Celsius for one hour.During incubation, prepare the necessary reagents. After incubation, wash the cells three times with DEPC PBS in a six-well plate for five minutes each. Then, wash the cells with FISH wash buffer three times for five minutes each.And keep the cover slip in FISH wash buffer until the secondary antibody, an intron probe mixture has been prepared. Next, clean the glass slides for primary antibody incubation for hybridization with soapy water. After they're dry, clean them with ethanol and a lab tissue.Place 40 microliters of the secondary antibody and FISH intron probe with solution onto each glass slide. Place the cover slips with the cells onto the slides with the cells side facing down on top of the buffer. And be careful not to introduce bubbles.Place the slides into a plastic container with a moist paper towel on the bottom. Wrap the plastic container in first plastic wrap, and then aluminum foil. And incubate the container with the slides.After incubation, carefully slide the cover slip from the edge of the glass slide and place it back into six-well plates, facing up. Wash the cells with FISH wash buffer three times in a six-well plate for five minutes each. Next, wash the cells two times with two-fold SSC.Add DAPI at one to 1000 concentration into the two-fold SSC and allow the solution to sit for five minutes at room temperature. Wash the cells twice with two-fold SSC. Clean the glass slides used for hybridization and add 10 microliters of mounting solution onto the glass slides.Place the cover slips face down onto the mounting solution. With a laboratory tissue, gently remove the excess mounting solution, being careful not to squash the cells. Using nail polish, apply an ample layer around the edge of the cover slips and allow the polish to dry.The sample is now ready for fluorescence microscopy. BCBL-1 cells were stained for LANA and K-Rta RNA to examine where actively transcribing viral episomes are and the heterogeneity of response to Kaposi's Sarcoma-associated Herpesvirus, or KSHV reactivation stimuli. The distinct K-Rta fluorescence in regions that closely match the distribution of viral episomes is evidence for active transcription taking place close to KSHV genomes.In the same sample, cells can be observed which exhibit much weaker and diffuse K-Rta fluorescence that does not significantly overlap with LANA, illustrating the considerable variation and the degree of response to reactivation stimuli within a population of cells. Next, the effectiveness of thymidine synchronization was examined by visualizing the expression of viral protein which clearly demonstrated that synchronized cells responded more to the reactivation stimuli than the unsynchronized population. DAPI staining can serve several functions, this example clearly shows that there are three separate cells instead of one.3D microscopy alongside this technique revealed ring-like RNA PII structures seen with KSHV genomes dotted on the periphery. With its development, the mini viral chromosome would provide unique tools for researchers to explore the special temporal organization of gene-expression and the regression of the cell nucleus.

Research

JoVE Journal

Biology

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

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

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 ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal ...

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...