We are grateful to the staff of the advanced imaging facility of CIBIO at the University of Trento and the BioOptics Light Microscopy facility at the Max F. Perutz Laboratories (MFPL) in Vienna. The research leading to these results has received funding from the Mahlke-Obermann Stiftung and the European Union&#39;s Seventh Framework Programme for research, technological development and demonstration under grant agreement no 609431 to EC. EC is supported by a Rita Levi Montalcini fellowship from the Italian Ministry of Education University and Research (MIUR).

1 . Selection of Neomycin Resistant Clones
<ol>
	<li>Grow AGS cells in Ham&#39;s F-12K (Kaighn&#39;s) medium supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine, penicillin (0.5 units per mL of medium), and streptomycin (0.2 &#181;g per mL of medium) at 37 &#176;C and 5% CO2. Transfect the cells at a 50-60% confluence with the sgRNA/Cas9 expressing vector and the MS2 cassette at a 1:10 molar ratio21. 
	NOTE: In a parallel experiment, verify transfection efficiency by...

<ol>
	<li>Azzalin, C. M., Reichenbach, P., Khoriauli, L., Giulotto, E., Lingner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeric+repeat+containing+RNA+and+RNA+surveillance+factors+at+mammalian+chromosome+ends.">Telomeric repeat containing RNA and RNA surveillance factors at mammalian chromosome ends.</a> Science. 318 (5851), 798-801 (2007).</li><li>Schoeftner, S., Blasco, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmentally+regulated+transcription+of+mammalian+telomeres+by+DNA-dependent+RNA+polymerase+II.">Developmentally regulated transcription of mammalian telomeres by DNA-dependent RNA polymerase II.</a> Nature Cell Biology. 10 (2), 228-236 (2008).</li><li>Diman, A., Decottignies, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+origin+and+nuclear+localization+of+TERRA+telomeric+repeat-containing+RNA:+from+Darkness+to+Dawn.">Genomic origin and nuclear localization of TERRA telomeric repeat-containing RNA: from Darkness to Dawn.</a> FEBS Journal. , (2017).</li><li>Arnoult, N., Van Beneden, A., Decottignies, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomere+length+regulates+TERRA+levels+through+increased+trimethylation+of+telomeric+H3K9+and+HP1alpha.">Telomere length regulates TERRA levels through increased trimethylation of telomeric H3K9 and HP1alpha.</a> Nature Structural &amp; Molecular Biology. 19 (9), 948-956 (2012).</li><li>Montero, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TERRA+recruitment+of+polycomb+to+telomeres+is+essential+for+histone+trymethylation+marks+at+telomeric+heterochromatin.">TERRA recruitment of polycomb to telomeres is essential for histone trymethylation marks at telomeric heterochromatin.</a> Nature Communications. 9 (1), 1548 (2018).</li><li>Beishline, K., 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=CTCF+driven+TERRA+transcription+facilitates+completion+of+telomere+DNA+replication.">CTCF driven TERRA transcription facilitates completion of telomere DNA replication.</a> Nature Communications. 8 (1), 2114 (2017).</li><li>Arora, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNaseH1+regulates+TERRA-telomeric+DNA+hybrids+and+telomere+maintenance+in+ALT+tumour+cells.">RNaseH1 regulates TERRA-telomeric DNA hybrids and telomere maintenance in ALT tumour cells.</a> Nature Communications. 5, 5220 (2014).</li><li>Balk, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeric+RNA-DNA+hybrids+affect+telomere-length+dynamics+and+senescence.">Telomeric RNA-DNA hybrids affect telomere-length dynamics and senescence.</a> Nature Structural &amp; Molecular Biology. 20 (10), 1199-1205 (2013).</li><li>Graf, 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=Telomere+Length+Determines+TERRA+and+R-Loop+Regulation+through+the+Cell+Cycle.">Telomere Length Determines TERRA and R-Loop Regulation through the Cell Cycle.</a> Cell. 170 (1), 72-85 (2017).</li><li>Lee, Y. W., Arora, R., Wischnewski, H., Azzalin, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRF1+participates+in+chromosome+end+protection+by+averting+TRF2-dependent+telomeric+R+loops.">TRF1 participates in chromosome end protection by averting TRF2-dependent telomeric R loops.</a> Nature Structural &amp; Molecular Biology. , (2018).</li><li>Moravec, 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=TERRA+promotes+telomerase-mediated+telomere+elongation+in+Schizosaccharomyces+pombe.">TERRA promotes telomerase-mediated telomere elongation in Schizosaccharomyces pombe.</a> EMBO Reports. 17 (7), 999-1012 (2016).</li><li>Cusanelli, E., Romero, C. A., Chartrand, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeric+noncoding+RNA+TERRA+is+induced+by+telomere+shortening+to+nucleate+telomerase+molecules+at+short+telomeres.">Telomeric noncoding RNA TERRA is induced by telomere shortening to nucleate telomerase molecules at short telomeres.</a> Molecular Cell. 51 (6), 780-791 (2013).</li><li>Moradi-Fard, 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=Smc5/6+Is+a+Telomere-Associated+Complex+that+Regulates+Sir4+Binding+and+TPE.">Smc5/6 Is a Telomere-Associated Complex that Regulates Sir4 Binding and TPE.</a> PLoS Genetics. 12 (8), e1006268 (2016).</li><li>Chu, H. 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=TERRA+RNA+Antagonizes+ATRX+and+Protects+Telomeres.">TERRA RNA Antagonizes ATRX and Protects Telomeres.</a> Cell. 170 (1), 86-101 (2017).</li><li>Lai, L. T., Lee, P. J., Zhang, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+protects+RNA+signals+in+simultaneous+RNA-DNA+FISH.">Immunofluorescence protects RNA signals in simultaneous RNA-DNA FISH.</a> Experimental Cell Research. 319 (3), 46-55 (2013).</li><li>Zhang, L. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeric+RNAs+mark+sex+chromosomes+in+stem+cells.">Telomeric RNAs mark sex chromosomes in stem cells.</a> Genetics. 182 (3), 685-698 (2009).</li><li>Gallardo, F., Chartrand, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+mRNAs+in+fixed+and+living+yeast+cells.">Visualizing mRNAs in fixed and living yeast cells.</a> Methods in Molecular Biology. 714, 203-219 (2011).</li><li>Querido, E., Chartrand, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+fluorescent+proteins+to+study+mRNA+trafficking+in+living+cells.">Using fluorescent proteins to study mRNA trafficking in living cells.</a> Methods in Cell Biology. 85, 273-292 (2008).</li><li>Cusanelli, E., Chartrand, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeric+noncoding+RNA:+telomeric+repeat-containing+RNA+in+telomere+biology.">Telomeric noncoding RNA: telomeric repeat-containing RNA in telomere biology.</a> Wiley Interdisciplinary Reviews: RNA. 5 (3), 407-419 (2014).</li><li>Perez-Romero, C. A., Lalonde, M., Chartrand, P., Cusanelli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+relocalization+of+telomeric+repeat-containing+RNAs+during+diauxic+shift+in+budding+yeast.">Induction and relocalization of telomeric repeat-containing RNAs during diauxic shift in budding yeast.</a> Current Genetics. , (2018).</li><li>Avogaro, L., 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=Live-cell+imaging+reveals+the+dynamics+and+function+of+single-telomere+TERRA+molecules+in+cancer+cells.">Live-cell imaging reveals the dynamics and function of single-telomere TERRA molecules in cancer cells.</a> RNA Biology. , 1-10 (2018).</li><li>Ran, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+nicking+by+RNA-guided+CRISPR+Cas9+for+enhanced+genome+editing+specificity.">Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.</a> Cell. 154 (6), 1380-1389 (2013).</li><li>Yamada, T., 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=Spatiotemporal+analysis+with+a+genetically+encoded+fluorescent+RNA+probe+reveals+TERRA+function+around+telomeres.">Spatiotemporal analysis with a genetically encoded fluorescent RNA probe reveals TERRA function around telomeres.</a> Scientific Reports. 6, 38910 (2016).</li><li>Deng, Z., 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=Formation+of+telomeric+repeat-containing+RNA+(TERRA)+foci+in+highly+proliferating+mouse+cerebellar+neuronal+progenitors+and+medulloblastoma.">Formation of telomeric repeat-containing RNA (TERRA) foci in highly proliferating mouse cerebellar neuronal progenitors and medulloblastoma.</a> Journal of Cell Science. 125 (Pt 18), 4383-4394 (2012).</li><li>Casini, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+specific+SpCas9+variant+is+identified+by+in+vivo+screening+in+yeast.">A highly specific SpCas9 variant is identified by in vivo screening in yeast.</a> Nature Biotechnology. 36 (3), 265-271 (2018).</li><li>Nergadze, S. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomerase+efficiently+elongates+highly+transcribing+telomeres+in+human+cancer+cells.">Telomerase efficiently elongates highly transcribing telomeres in human cancer cells.</a> PLoS One. 7 (4), e35714 (2012).</li><li>Flynn, R. 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In this article we present a method to generate human cancer cell clones containing MS2 sequences integrated within subtelomere 15q. Using these clones, the MS2-tagged TERRA molecules transcribed from the subtelomere 15q are detected by fluorescence microscopy by co-expression of a MS2-GFP fusion protein. This approach enables researchers to study the dynamics of TERRA expressed from a single telomere in living cells21. In this protocol, TERRA-MS2 clones are selected in the AGS cell line, which re...

The long noncoding RNA TERRA is transcribed from the subtelomeric region of chromosomes and its transcription proceeds towards the chromosome ends, terminating within the telomeric repeat tract1,2. For this reason, TERRA transcripts consist of subtelomeric-derived sequences at their 5&#39; end and terminate with telomeric repeats (UUAGGG in vertebrates)3. Important roles have been proposed for TERRA, including heterochromatin formation at telomeres4,5, DNA replication6, promoting homologous recombination among chromosome ends7,8,9, regulating telomere structure10and telomere length homeostasis2,11,12,13. Furthermore, TERRA transcripts interact with numerous extratelomeric sites to regulate widespread gene expression in mouse embryonic stem (ES) cells14. In line with these evidence, RNA fluorescence in situ hybridization (RNA-FISH) analyses have shown that only a subset of TERRA transcripts localize at telomeres1,2,15. In addition, TERRA has been reported to form nuclear aggregates localizing at the X and Y chromosomes in mouse cells2,16. These findings indicate that TERRA transcripts undergo complex dynamics within the nucleus. Understanding the dynamics of TERRA molecules will help define their function and mechanisms of action.
The MS2-GFP system has been widely used to visualize RNA molecules in living cells from various organisms17,18. This system has been previously used to tag and visualize single-telomere TERRA molecules in S. cerevisiae12,19. Using this system, it was recently shown that yeast TERRA transcripts localize within the cytoplasm during the post-diauxic shift phase, suggesting that TERRA may exert extranuclear functions20. We have recently used the MS2-GFP system to study single-telomere TERRA transcripts in cancer cells21. To this aim, we employed the CRISPR/Cas9 genome editing tool to integrate MS2 sequences at a single telomere (telomere 15q, hereafter Tel15q) and obtained clones expressing MS2-tagged endogenous Tel15q TERRA (TERRA-MS2 clones). Co-expression of a GFP-fused MS2 RNA-binding protein (MS2-GFP) that recognizes and binds MS2 RNA sequences enables visualization of single-telomere TERRA transcripts in living cells21. The purpose of the protocol illustrated here is to describe in detail the steps required for the generation of TERRA-MS2 clones.
To generate TERRA-MS2 clones, a MS2 cassette is integrated within the subtelomeric region of telomere 15q, downstream of the TERRA promoter region and transcription start site. The MS2 cassette contains a neomycin resistance gene flanked by lox-p sites, and its integration at subtelomere 15q is performed using the CRISPR/Cas9 system22. After transfection of the MS2 cassette, single clones are selected and subtelomeric integration of the cassette is verified by PCR, DNA sequencing and Southern blot. Positive clones are infected with a Cre-expressing adenovirus in order to remove the selection marker in the cassette, leaving only MS2 sequences and a single lox-p site at the subtelomere 15q. Expression of MS2-tagged TERRA transcripts from Tel15q is verified by RT-qPCR. Finally, the MS2-GFP fusion protein is expressed in TERRA-MS2 clones via retroviral infection in order to visualize MS2-TERRA transcripts by fluorescence microscopy. TERRA transcripts can be readily detected by RNA-FISH and live-cell imaging using telomeric repeat-specific probes1,2,15,23. These approaches provide important information on the localization of the total population of TERRA molecules at single cell resolution. The generation of clones containing MS2 sequences at a single subtelomere will enable researchers to study the dynamics of single-telomere TERRA transcripts in living cells, which will help define the function and mechanisms of action of TERRA.

<table border="0" cellpadding="0" cellspacing="0" style="width:1177px;" width="1178">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:245px;">AGS cells</td>
			<td style="width:246px;">-</td>
			<td style="width:105px;">-</td>
			<td style="width:583px;">Gift from Christian Baron (Universit&#233; de Montr&#233;al).</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">F12K Nut Mix 1X</td>
			<td>GIBCO</td>
			<td>21127022</td>
			<td>Culturing medium for AGS cells</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">L-Glutamine&#160;</td>
			<td>CORNING</td>
			<td>MT25005CI</td>
			<td>Component of cell culturing medium</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Penicillin Streptomycin Solution</td>
			<td>CORNING</td>
			<td>30-002-CI</td>
			<td>Component of cell culturing medium</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Fetal Bovine Serum</td>
			<td>Sigma Aldrich</td>
			<td>F2442</td>
			<td>Component of cell culturing medium</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DMEM 1X</td>
			<td>GIBCO</td>
			<td>21068028</td>
			<td>culturing medium for phoenix cell</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">CaCl2</td>
			<td>Sigma Aldrich</td>
			<td>C1016</td>
			<td>used in phoenix cell transfection</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H3375</td>
			<td>used in phoenix cell transfection (HBS solution)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">KCl</td>
			<td>Sigma Aldrich</td>
			<td>P9333</td>
			<td>used in phoenix cell transfection (HBS solution)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Dextrose</td>
			<td>Sigma Aldrich</td>
			<td>D9434</td>
			<td>used in phoenix cell transfection (HBS solution)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">NaCl</td>
			<td>Sigma Aldrich</td>
			<td>S7653</td>
			<td>used in phoenix cell transfection (HBS solution) and retrovirus precipitation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Na2HPO4</td>
			<td>Sigma Aldrich</td>
			<td>S3264</td>
			<td>used in phoenix cell transfection (HBS solution)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">TRYPSIN EDTA SOLUTION 1X</td>
			<td>CORNING</td>
			<td>59430C</td>
			<td>used in cell split</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DPBS 1X</td>
			<td>GIBCO</td>
			<td>14190250</td>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DMSO</td>
			<td>Sigma Aldrich</td>
			<td>D8418</td>
			<td>Component of cell freezing medium (80% FBB and 20% DMSO)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:245px;">G-418 Disulphate</td>
			<td>Formedium</td>
			<td>G4185</td>
			<td>selection drug for&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Gelatin solution Bioreagent</td>
			<td>Sigma Aldrich</td>
			<td>G1393</td>
			<td>cotaing of 96 well DNA plate and freezing plate</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Tris-base</td>
			<td>Fisher BioReagents</td>
			<td>10376743</td>
			<td>Component of Cell lysis buffer for genomic DNA extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">EDTA</td>
			<td>Sigma Aldrich</td>
			<td>E6758</td>
			<td>Component of Cell lysis buffer for genomic DNA extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">SDS</td>
			<td>Sigma Aldrich</td>
			<td>71729</td>
			<td>Component of Cell lysis buffer for genomic DNA extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Proteinase K</td>
			<td>Thermo Fisher</td>
			<td>AM2546</td>
			<td>Component of Cell lysis buffer for genomic DNA extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">RNAse A</td>
			<td>Thermo Fisher</td>
			<td>12091021</td>
			<td>RNA degradation during DNA extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Agarose</td>
			<td>Sigma Aldrich</td>
			<td>A5304</td>
			<td>DNA gel preparation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Atlas ClearSight</td>
			<td>Bioatlas</td>
			<td>BH40501</td>
			<td>Stain reagent used for detecting DNA and RNA samples in agarose gel</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">ethanol</td>
			<td>Fisher BioReagents</td>
			<td>BP28184</td>
			<td>DNA precipitation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Sodium Acetate&#160;</td>
			<td>Sigma Aldrich</td>
			<td>71196</td>
			<td>Used for DNA precipitation at a 3M concentration pH5.2</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Wizard SV Gel and PCR clean-Up system</td>
			<td>Promega</td>
			<td>A9282</td>
			<td>Extraction of PCR fragments from agarose gel during PCR screening of neomycin positive clones</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Trizol</td>
			<td>AMBION</td>
			<td>15596018</td>
			<td>Organic solvent used for RNA extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Dnase I</td>
			<td>THERMO SCIENTIFIC</td>
			<td>89836</td>
			<td>degradation of genomic DNA from RNA&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">dNTPs mix</td>
			<td>Invitrogen</td>
			<td>10297018</td>
			<td>used in RT and PCR reactions</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DTT</td>
			<td>Invitrogen</td>
			<td>707265ML</td>
			<td>used in RT reactions</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">diethyl pyrocarbonate</td>
			<td>Sigma Aldrich</td>
			<td>D5758</td>
			<td>used to inactivate RNAses in water (1:1000 dilution)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ribolock</td>
			<td>Thermo Fisher</td>
			<td>EO0381</td>
			<td>RNase inhibitor</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MOPS</td>
			<td>Sigma Aldrich</td>
			<td>M9381</td>
			<td>preparation of RNA gel</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>preparation of denaturating RNA gel (1% PFA in 1x MOPS)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Superscript III Reverse transcriptase</td>
			<td>Invitrogen</td>
			<td>18080-093</td>
			<td>Retrotranscription reaction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Pfu DNA polymerase (recombinant)</td>
			<td>Thermo Scientific</td>
			<td>EP0501</td>
			<td>PCR reaction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">2X qPCRBIO SyGreen Mix Separate-ROX</td>
			<td>PCR BIOSYSTEMS</td>
			<td>PB 20.14</td>
			<td>qPCR reaction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Cre-GFP adenovirus</td>
			<td>https://medicine.uiowa.edu/vectorcore</td>
			<td>1174-HT</td>
			<td>used to infect TERRA-MS2 clones in order to remove the neomycn gene</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Sodium Butyrate</td>
			<td>Sigma Aldrich</td>
			<td>B5887</td>
			<td>used to promote retrovirus particles production in phoenix cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PEG8000</td>
			<td>Sigma Aldrich</td>
			<td>89510</td>
			<td>Precipitation of retrovirus partcles</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">35&#181;-Dish Glass Bottom</td>
			<td>Ibidi</td>
			<td>81158</td>
			<td>used in live cell imaging analyses of TERRA-MS2 clones</td>
		</tr>
	</tbody>
</table>

generation of cancer cell clones to visualize telomeric repeat-containing rna terra expressed from a single telomere in living cells

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. Recent findings revealed that TERRA molecules also interact with internal chromosomal regions to regulate gene expression in mouse embryonic stem (ES) cells. In line with this evidence, RNA fluorescence in situ hybridization (RNA-FISH) analyses have shown that only a subset of TERRA transcripts localize at chromosome ends. A better understanding of the dynamics of TERRA molecules will help define their function and mechanisms of action. Here, we describe a method to label and visualize single-telomere TERRA transcripts in cancer cells using the MS2-GFP system. To this aim, we present a protocol to generate stable clones, using the AGS human stomach cancer cell line, containing MS2 sequences integrated at a single subtelomere. Transcription of TERRA from the MS2-tagged telomere results in the expression of MS2-tagged TERRA molecules that are visualized by live-cell fluorescence microscopy upon co-expression of a MS2 RNA-binding protein fused to GFP (MS2-GFP). This approach enables researchers to study the dynamics of single-telomere TERRA molecules in cancer cells, and it can be applied to other cell lines.

Figure 1 represents an overview of the experimental strategy. The main steps of the protocol and an indicative timeline for the generation of TERRA-MS2 clones in AGS cells are shown (Figure 1A). At day 1, multiple wells of a 6 well plate are transfected with the MS2 cassette and sgRNA/Cas9 expressing vectors (shown in Figure 1B). Two different subtelomere...

Watch this Scientific Journal Video about Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells at JoVE.com

Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells

Here, we present a protocol to generate cancer cell clones containing a MS2 sequence tag at a single subtelomere. This approach, relying on the MS2-GFP system, enables visualization of the endogenous transcripts of telomeric repeat-containing RNA (TERRA) expressed from a single telomere in living cells.

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in ...

This method can help answer key questions about Telomeres, such as yeast telomeric noncoding RNA TERRA localizing, and I can insist that it's telomere of origin or in trans, a different cromes-mants The main advantage of this technique is that it allows researchers to visualize TERRA molecules express from a single telomere in living cells. Demonstrating the procedure will be Claudio Oss Pegorar and Nicole Bettin, two graduate students from my laboratory. To begin grow AGS cells according to the text protocol.Then when the cells reach 50 to 60%confluence transpect them with sgRNA Cas9 expressing vector and the MS2 cassette. The next day replace the culturing medium with medium containing Neomycin. After this add 10 microliters of 0.25%Tripsyn to each well of a 96 well plate.Using a microscope and a marker, mark the position of each clone visible in the dish. Replace the Neomycin culturing medium with enough PBS to form a thin film of liquid on the clones. Using a 10 microliter pipette containing five microliters of Trypsin, slowly release the Trypsin onto the colony.Allow the Trypsin to detach the cells for one minute. Scrape the colony with the tip, and suck it up into the tip. After this, transfer the cells from the colony into a well of the 96 well plate.Incubate the plate for five minutes at room temperature. Then fill the well with 150 microliters of selective medium. Next, add 100 microliters of gelatin to each well of three 96 well plates.Incubate the plates at room temperature for 30 minutes, and wash the plates with PBS twice. After the clones reach 90%confluence, aspirate the medium from each well of the plate, and wash the plate with PBS. Then add 30 microliters of Trypsin to each well, and incubate the plate at 37 degrees celsius for five minutes.Next, add 70 microliters of selective medium to each of the wells. Disrupt the cells by pipetting up and down. Transfer 30 microliters of the mixture to the well of the gelatinized DNA plate containing 120 microliters of selective medium.Then transfer 30 microliters of the mixture to the gelatinized freezing plate containing 50 microliters of medium. Place the DNA in the incubator, and allow the cells to grow at 37 degrees celsius until they reach 90%confluence. After this, add 80 microliters of ice cold freezing medium to each well of the freezing plate.Seal the plate with parafilm, replace the lid, and wrap the plate in aluminum foil. Then keep the freezing plates at 80 degrees celsius. After the clones in the DNA plate reach 90%confluence, wash the plate with PBS twice.Then lyse the cells with 50 microliters of Lysis buffer. Cover the plate with parafilm, replace the lid, and wrap the plate in saran wrap. After this, incubate the plate at 37 degrees celsius overnight.The next day add 100 microliters of ethanol sodium chloride solution to each well, and allow DNA precipitation for six hours or overnight at room temperature. Then invert the plate and wash it three times with 200 microliters of ethanol 70%per well. Add 20 microliters of RNase A solution to each well of the plate, and incubate the plate at 37 degrees celsius for one hour.After this, use three microliters of genomic DNA for PCR amplification, and setup the PCR machine. Grow the PCR positive clones in six well plates according to the text protocol. Once the clones reach 90%confluence, wash the cells with PBS, and add 250 microliters of Lysis buffer with Proteinase K to each well.Use a cell scraper to scrape the cells, and transfer the Lysate to a 1.5 milliliter tube. Incubate the Lysate at 37 degrees celsius for 16 hours. Add one milliliter of ethanol to the Lysate, and shake vigorously.Allow the clones to grow in F-12K medium according to the text protocol. Then add the Cre-expressing adenovirus to the cells once they reach 70%confluence. 48 hours after infection, split the cells into three 10 centimeter dishes.Then culture the third dishes for cell freezing, and RNA extraction. First, plate the TERRA-MS2 clones and the wild type AGS cells in glass bottom dishes. Then add Polybrene and the MS2-GFP expressing retrovirus to the medium.After 24 hours discard the medium containing the retrovirus, and add fresh medium without phenol red to the cells. Finally, using an inverted microscope and a sensitive camera image the cells with the appropriate objective and aperture. In this protocol cancer cell clones containing an MS2 sequence tag at a single subtelomere were created and imaged.The Neomycin resistant clones were screened using PCR. The positive clones were cultured at lysed for genomic DNA extraction and Southern blot analysis. The clones positive to PCR and Southern blot analysis were infected with the CRE-GFP expressing Adenovirus in order to remove the Neomycin gene present in the MS2 cassette.Once the elimination of the Neomycin resistance gene was confirmed, RNA extracted from the clones were tested for the expression of TERRA-MS2 transcripts. In order to visualize TERRA-MS2 transcripts in living cells by confocal microscopy, the selected clones were infected with a retrovirus expressing an MS2-GFP fusion protein. TERRA-MS2 transcripts and telomeres were simultaneously visualized in living cells via confocal microscopy by co-expressing the MS2-GFP fusion protein and an mCherry fused telomere binding protein, TRF2, in the selected clones.When attempting this procedure it's important to make every effort to select a single clone instead of a mixed population of clones. Following this procedure, other methods like a DNA FISH on chromosome spreads, can be used in order to visualize the integration of the MS2 cassette in fixed cells. After its development, this technique may pave the way for researchers in the field of telomeres to explore the cellular localization of single telomere TERRA molecules in living human cells.Don't forget that working with viral vectors and radioactive materials can be extremely uncertain. And precautions, such as wearing lab coats, gloves and using plexiglass shields to minimize radiation should always be taken when performing this procedure.

generation-cancer-cell-clones-to-visualize-telomeric-repeat

Research

JoVE Journal

Genetics

7.3K Aufrufe.  University of Trento. Diese Methode kann helfen, wichtige Fragen über Telomere zu beantworten, wie Hefe telomernichtcodierende RNA TERRA Lokalisierung, und ich kann darauf bestehen, dass es Telomer des Ursprungs oder in trans, ein anderes cromes-mants Der Hauptvorteil dieser Technik ist, dass es Forschern ermöglicht, TERRA Moleküle aus einem einzigen Telomer in lebenden Zellen auszudrücken. Demonstriert wird das Verfahren von Claudio Oss Pegorar und Nicole Bettin, zwei Doktoranden aus meinem Labor. Um mit dem ...