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 transfecting a GFP-expressing vector (i.e., Cas9-GFP vector). At least 60-70% transfection efficiency should be achieved.</li>
	<li>The following day, replace the culturing medium with medium containing neomycin at 0.7 &#181;g/mL final concentration (selective medium). 
	NOTE: Splitting the cells and seeding them in selective medium the day after transfection will speed up the selection process. All non-transfected cells will immediately die. If transfection is performed in 6 well plates, in which case each well at a 60% confluence would contain approximately 0.7 x 106&#160;cells, on the following day the cells can be split from a single well to a 10 cm dish containing selective medium.</li>
	<li>Keep the cells in selective medium for 7-10 days, changing medium every one or two days, until single clones are visible.</li>
	<li>Picking of cell clones
	<ol>
		<li>Prepare a 96 well plate containing 10 &#181;L of 0.25% trypsin in each well. 
		NOTE: Prepare two 96 well plates in case more than 96 clones are expected to be picked.</li>
		<li>With the use of a microscope, mark the position of each clone visible in the 10 cm dish by making a dot at the bottom of the dish using a marker. Each dot will correspond to a colony to be picked.</li>
		<li>Replace the culturing medium with just enough Phosphate Buffered Saline (PBS) to form a thin film of liquid on the clones and not let the cells dry during the clone picking.</li>
		<li>Pick single colonies using a 10 &#181;L pipette. Attach the tip containing 5 &#181;L of trypsin to the colony and slowly release the trypsin which will remain localized on the colony. Allow trypsin to detach the cells for 1 min, then scrape the colony with the tip and suck it up into the tip. 
		NOTE: During this procedure, flipping the dish a bit on one side so to decrease the volume of PBS around the colony being picked will help the picking process by avoiding diluting the trypsin around the colony. Using a clone ring may also help picking up single clones.</li>
		<li>Place the cells from the colony into a well of the 96 well plate containing 10 &#181;L of 0.25% trypsin.</li>
		<li>Incubate 5 min at room temperature, then fill the well with 150 &#181;L of selective medium (Ham&#39;s F-12K (Kaighn&#39;s) medium supplemented with 10% FBS, 2 mM L-glutamine, penicillin (0.5 units per mL of medium), streptomycin (0.2 &#181;g per mL of medium) and containing neomycin at a concentration of 0.7 &#181;g/mL. 
		NOTE: During the incubation time other clones can be picked. It is recommended to pick as many clones as possible. The more clones are picked the higher the chances will be of identifying positive ones.</li>
		<li>Once all the clones are picked and transferred in the 96 well plate, allow the cells to grow for a few days in selective medium Ham&#39;s F-12K (Kaighn&#39;s) medium supplemented with 10% FBS, 2 mM L-glutamine, penicillin (0.5 units per mL of medium), streptomycin (0.2 &#181;g per mL of medium) and containing neomycin at a concentration of 0.7 &#181;g/mL&#160;at 37 &#176;C and 5% CO2, until reaching 90% confluence.</li>
	</ol>
	</li>
	<li>Splitting of clones
	<ol>
		<li>Prepare three 96 well plates coated with gelatin by adding 100 &#181;L of gelatin per well, incubate for 30 min at room temperature, then wash two times with PBS. These plates will be used for DNA extraction (DNA plate) and for clone freezing (freezing plates). 
		NOTE: Gelatin will promote the attachment of the cells and of the DNA to the wells. In particular, the gelatin coating will allow the DNA to stick at the bottom of the wells during the DNA extraction and wash procedures (discussed below). During the clone-splitting procedure, it is advisable to use a multichannel pipette.</li>
		<li>Once clones reach 90% confluence, aspirate medium from each well of the 96 well plate, wash with PBS, add 30 &#181;L of 0.25% trypsin per well, and incubate for 5 min at 37 &#176;C. 
		NOTE: Clones will grow at different rates, which will also depend on the number of cells picked per clone. Thus, this step will be performed during the several days when the different clones reach 90% confluence.</li>
		<li>Add 70 &#181;L of selective medium per well, disrupt cell clumps by pipetting up and down inside the wells, then transfer 30 &#181;L of the 100 &#181;L to the gelatinized DNA plate prefilled with 120 &#181;L of selective medium per well and 30 &#181;L to the gelatinized freezing plates prefilled with 50 &#181;L medium (without selection).</li>
		<li>Place the DNA plate in the incubator and allow the cells to grow at 37 &#176;C and 5% CO2 until 90% confluence.</li>
		<li>Add 80 &#181;L of ice-cold freshly made 2x freezing medium (80% FBS and 20% dimethyl sulfoxide (DMSO)) to each well of the freezing plate, add parafilm (sprayed with 70% ethanol) on top of the plate so to seal each well, place the lid on top and wrap the plate with aluminum foil. Place the freezing plates at -80 &#176;C.</li>
		<li>Add 90 &#181;L of selective medium per well to the original 96 well plate containing the clones that have been split and keep it in culture until PCR screening results. This plate will be used as backup plate.</li>
	</ol>
	</li>
</ol>
2. Screening of Neomycin Resistant Clones
<ol>
	<li>DNA extraction from the 96 well DNA plate.
	<ol>
		<li>Once the clones cultured in the DNA plate reach 90% confluence, wash 2 times with PBS, then lyse with 50 &#181;L lysis buffer (10 mM Tris pH 7.5, 10 mM EDTA, 10 mM NaCl, 0.5% SDS, and 1 mg/mL proteinase K). Cover the plate with parafilm, sealing each well, put the lid on, cover with saran wrap, and place at 37 &#176;C overnight.</li>
		<li>Add 100 &#181;L of cold ethanol (Et-OH)/NaCl solution (0.75 M NaCl in 100% ethanol) to each well and precipitate for 6 h or overnight at room temperature. 
		NOTE: The protocol can be paused here.</li>
		<li>Remove the Et-OH/NaCl solution by inverting the plate and wash 3 times with 200 &#181;L of 70% ethanol per well.</li>
		<li>Add 25 &#181;L of RNAse A solution in distilled water and incubate at 37 &#176;C for 1 h.</li>
	</ol>
	</li>
	<li>Use 3 &#181;L of genomic DNA for PCR amplification. Perform PCR screening of the selected clones using primers annealing within the neomycin resistance gene and subtelomere 15q. PCR amplification is performed using standard polymerase enzymes and PCR protocols21. 
	NOTE: PCR conditions for MS2 primers (MS2-subtel15q-primer-S and MS2 primer AS) and CTR primers (CTR prime S and CTR primer AS) are the following: 98 &#176;C for 20 s as denaturation step and then 34 cycles at 98 &#176;C 10 s, 58&#176;C 20 s, 72 &#176;C 15 s, using polymerase enzyme in a 25 &#181;L reaction mix (see Table of Materials). Primer sequences are indicated in Table 1.</li>
	<li>Run PCR reactions on agarose gel and extract PCR bands obtained from positive clones using standard gel extraction procedures (see Table of Materials for gel extraction reagents).</li>
	<li>Perform DNA sequencing analyses of the gel-extracted PCR product for confirmation of the presence of the MS2 sequences21. 
	NOTE: The sequencing analyses can be performed using the primers used for the PCR screening.</li>
	<li>Southern blot screening of PCR positive clones
	<ol>
		<li>Grow the clones positive at PCR and sequencing screening from the original 96 well plate (the backup plate) to 6 well plates in Ham&#39;s F-12K (Kaighn&#39;s) medium supplemented with 10% FBS, 2 mM L-glutamine, penicillin (0.5 units per mL of medium), streptomycin (0.2 &#181;g per mL of medium) and containing neomycin at a concentration of 0.7 &#181;g/mL&#160;at 37 &#176;C 5% CO2. 
		NOTE: Alternatively, if some of these clones have been lost, thaw them from one of the freezing plates (see protocol 2.6).</li>
		<li>Once the clones are at 90% confluence in the 6 well plate, wash the cells with PBS and add 250 &#181;L of lysis buffer containing 0.5 &#181;g proteinase K per well.</li>
		<li>Scrape the cells using a cell scraper and transfer the lysate in a 1.5 mL tube.</li>
		<li>Incubate at 37 &#176;C for 16 h.</li>
		<li>Add 1 mL of 100% ethanol, shake vigorously, and allow the DNA to precipitate at least 2 h or overnight at -20 &#176;C. 
		NOTE: The protocol can be paused here.</li>
		<li>Spin at 13,400 x g at 4 &#176;C for 10 min, discard the supernatant, and wash the pellets with 70% ethanol. Let the pellets air dry at room temperature. Alternatively, use a vacuum concentrator.</li>
		<li>Resuspend the DNA pellets in 50 &#181;L of distilled water containing RNAse A and incubate for 1 h at 37 &#176;C.</li>
		<li>Digest 5-10 &#181;g of genomic DNA using NcoI and BamHI restriction enzymes (two independent digestions) in 100 &#181;L reaction volume by incubating the digestion reactions at 37 &#176;C overnight.</li>
		<li>Run 4 &#181;L of the digestion on agarose gel (0.8% agarose) for complete digestion confirmation.</li>
		<li>Add 1/10 volume of sodium acetate 3 M solution pH 5.2 and 2 volumes of 100% ethanol to the restriction digestion reactions and incubate at least 2 h or overnight at -20&#176;C to precipitate DNA. 
		NOTE: The protocol can be paused here.</li>
		<li>Centrifuge at 13,400 x g at 4 &#176;C for 20 min, discard the supernatants, and wash the pellets with 70% ethanol. Allow the pellets to air dry at room temperature. Alternatively, use a vacuum concentrator.</li>
		<li>Resuspend pellets in 20 &#181;L of distilled water and load the digested DNA on a 0.8% agarose gel. 
		NOTE: For a better resolution of the digested DNA, prepare a gel at least 15 cm long and run overnight at low voltage ( &#820;30 volts). The electrophoresis set up should be optimized.</li>
		<li>The following day, stain the gel with a DNA labelling agent, such as ethidium bromide at 1 &#181;L/10 mL final concentration, for 30 min at room temperature and take a picture with a ruler close to the gel on a gel imaging instrument.</li>
		<li>Set the transfer of DNA to a nylon membrane and perform membrane hybridization with a MS2 sequence-specific probe using standard procedures21.</li>
		<li>Thaw the clones that are positive at PCR, DNA sequencing and Southern blot from one of the two freezing plates (see next step).</li>
	</ol>
	</li>
	<li>Thawing of clones
	<ol>
		<li>Prepare one 15 mL tube containing 5 mL of pre-warmed Ham&#39;s F-12K (Kaighn&#39;s) medium supplemented with 10% FBS, 2 mM L-glutamine, penicillin (0.5 units per mL of medium), and streptomycin (0.2 &#181;g per mL of medium) for each clone to be thawed.</li>
		<li>Remove one of the freezing plates from -80&#176; and add 100 &#181;L of pre-warmed F12K complete medium to the well containing the positive clone to be thawed. 
		NOTE: This procedure should be performed quickly and the 96 well plate should be placed on dry ice after each clone is thawed, in order to allow the other clones to remain frozen. This is particularly important if multiple clones need to be thawed from the same 96 well plate.</li>
		<li>Transfer the cells to the 15 mL tube containing 5 mL of medium and centrifuge at 800 x g for 5 min at room temperature.</li>
		<li>Aspirate the medium, resuspend the cells in 500 &#181;L of pre-warmed complete F12K medium, and transfer each clone to a single well of a 12 well plate.</li>
	</ol>
	</li>
	<li>Elimination of the neomycin resistance gene from the MS2 cassette integrated at subtelomere 15q.
	<ol>
		<li>Allow the clones to grow from a 12 well plate to a 10 cm dish in complete F12K medium. 
		NOTE: Neomycin should not be included in the medium unless otherwise indicated.</li>
		<li>Add the Cre-expressing adenovirus to the cells cultured in a 10 cm dish at 70% confluence in 10 mL of complete F12K medium. 
		NOTE: Using a Cre-GFP expressing adenovirus will allow evaluation of the efficiency of infection, which should approach 100%. The replication-defective adenovirus will be lost after few passages in culture.</li>
		<li>48 hours after infection, split the cells in three 10 cm dishes. Two dishes will be used for verification of the neomycin gene removal by negative selection, growing the cells in neomycin-containing medium (first dish), and by Southern blot (second dish).</li>
		<li>Culture the third dish containing the clone for cell freezing and RNA extraction.</li>
	</ol>
	</li>
</ol>
3. Verification of TERRA-MS2 Transcript Expression by RT-qPCR
<ol>
	<li>Upon verification of neomycin gene removal, perform total RNA extraction from TERRA-MS2 clones using organic solvents (phenol and guanidine isothiocynate solution)21.
	<ol>
		<li>Resuspend the RNA extracted from a 10 cm dish in 100 &#181;L of diethyl pyrocarbonate (DEPC) water.</li>
		<li>Run 3 &#181;L of RNA on a denaturating (1% formaldehyde-containing) 1x 3-(N-Morpholino) propanesulfonic acid (MOPS) gel in order to verify concentration and integrity of the RNA. Also analyse RNA concentration using a spectrophotometer.</li>
		<li>Treat 3 &#181;g of RNA with DNAse I using 1 unit of DNAse I enzyme in 60 &#181;L final reaction volume.</li>
		<li>Incubate the reaction for 1 h at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Reverse transcription reaction and qPCR analyses
	<ol>
		<li>Add the following components to a nuclease-free microcentrifuge tube: 2 &#181;L of a 1 &#181;M TERRA specific primer, 1 &#181;L of dNTPs mix (10 mM each), 6 &#181;L of DNAse I-treated RNA (corresponding to &#820;300ng RNA). Adjust the volume to 13 &#181;L with 4 &#181;L of DEPC water. 
		NOTE: For each RNA to be analysed, a second tube containing the same reagents but a reference specific primer, instead of TERRA specific primer, should be prepared.</li>
		<li>Heat the mixture to 65 &#176;C for 5 min and incubate in ice for at least 1 min.</li>
		<li>Collect the content of the tubes by brief centrifugation and add 4 &#181;L of 5X RT enzyme Buffer, 1 &#181;L 0.1 M dithiothreitol (DTT), 1 &#181;L (4 units) of RNAse inhibitor, and 1 &#181;L of reverse transcriptase (see Table of Materials).</li>
		<li>Incubate the samples at 42 &#176;C for 60 min, then use 2 &#181;L of the RT reaction for qPCR analyses.</li>
	</ol>
	</li>
	<li>Prepare qPCR reaction mix in a final volume of 20 &#181;L consisting of 10 &#181;L of 2x qPCR master mix, 2 &#181;L of cDNA template, 1 &#181;L of forward primer (10 &#181;M), 1 &#181;L of reverse primer (10 &#181;M), and 6 &#181;L of water.</li>
	<li>Perform qPCR reaction in a thermocycler using standard protocols21.</li>
</ol>
4. Production of a MS2-GFP Expressing Retrovirus
<ol>
	<li>To generate MS2-GFP expressing retrovirus, transfect 80% confluent phoenix packaging cells with a MS2-GFP fusion protein expressing retrovirus vector (pBabe-MS2-GFP PURO) and an env gene expressing vector (such as pCMV-VSVG) using a molar ratio 4:1 of the two vectors.</li>
	<li>On the following day, replace the culturing medium (DMEM supplemented with 10% FBS, 2 mM L-glutamine and Pen/Strep) with fresh medium containing 10mM sodium butyrate.</li>
	<li>Incubate for 8 h at 37 &#176;C, 5% CO2, then replace the medium with fresh culturing medium from which the virus will be collected.</li>
	<li>After 48 h, remove the retrovirus-containing medium from the phoenix cells. This medium can be directly used for infection of TERRA-MS2 clones, in which case filter the medium through a 0.45 &#181;m filter, add polybrene (30 &#181;g/mL final concentration) and add it to the cells (in this case the protocol continues at step 5). Alternatively, retrovirus can be precipitated in a 50 mL falcon tube by adding 1/5th volume of 50% PEG-8000/900 mM NaCl solution and incubating overnight at 4 &#176;C on a rotator wheel.</li>
	<li>On the following day, pellet retrovirus particles by centrifugation at 2,000 x g for 30 min, remove supernatant, and resuspend the pellet in F12K medium without serum. 
	NOTE: The virus particles can be resuspended in 1/100th of the original supernatant volume. An infection test should be performed in order to verify the minimum volume of retrovirus required to efficiently infect the cells.</li>
</ol>
5. Visualization of TERRA-MS2 Transcripts in Living Cells
<ol>
	<li>Plate TERRA-MS2 clones and WT AGS cells in glass-bottomed dishes. On the day of infection, add polybrene to the medium (30 &#181;g/mL final concentration) and the MS2-GFP expressing retrovirus.</li>
	<li>After 24 hours, discard the virus-containing medium and add fresh medium without phenol-red.</li>
	<li>Analyze the cells at an inverted microscope using the appropriate microscope setting. Image the cells with a 100X or 60X objective with large numerical aperture (1.4X) and using a sensitive camera (EMCCD). Use an environmental control system to maintain the samples at 37 &#176;C and 5% CO2&#160;during imaging.</li>
</ol>

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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. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CpG-island+promoters+drive+transcription+of+human+telomeres.">CpG-island promoters drive transcription of human telomeres.</a> RNA. 15 (12), 2186-2194 (2009).</li><li>Porro, 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=Functional+characterization+of+the+TERRA+transcriptome+at+damaged+telomeres.">Functional characterization of the TERRA transcriptome at damaged telomeres.</a> Nature Communications. 5, 5379 (2014).</li><li>Farnung, B. O., Brun, C. M., Arora, R., Lorenzi, L. E., 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=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. 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=Alternative+lengthening+of+telomeres+renders+cancer+cells+hypersensitive+to+ATR+inhibitors.">Alternative lengthening of telomeres renders cancer cells hypersensitive to ATR inhibitors.</a> Science. 347 (6219), 273-277 (2015).</li><li>Scheibe, 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=Quantitative+interaction+screen+of+telomeric+repeat-containing+RNA+reveals+novel+TERRA+regulators.">Quantitative interaction screen of telomeric repeat-containing RNA reveals novel TERRA regulators.</a> Genome Research. 23 (12), 2149-2157 (2013).</li><li>Bolland, D. J., King, M. R., Reik, W., Corcoran, A. E., Krueger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+3D+DNA+FISH+using+directly+labeled+probes.">Robust 3D DNA FISH using directly labeled probes.</a> Journal of Visualized Experiments. (78), (2013).</li><li>Masui, O., 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+chromosome+dynamics+and+outcome+of+X+chromosome+pairing+events+during+ES+cell+differentiation.">Live-cell chromosome dynamics and outcome of X chromosome pairing events during ES cell differentiation.</a> Cell. 145 (3), 447-458 (2011).</li></ol>

The authors declare no competing financial interests

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><tbody><tr><td>AGS cells</td><td>-</td><td>-</td><td>Gift from Christian Baron (Universit&amp;eacute; de Montr&amp;eacute;al).</td></tr><tr><td>F12K Nut Mix 1X</td><td>GIBCO</td><td>21127022</td><td>Culturing medium for AGS cells</td></tr><tr><td>L-Glutamine&amp;nbsp;</td><td>CORNING</td><td>MT25005CI</td><td>Component of cell culturing medium</td></tr><tr><td>Penicillin Streptomycin Solution</td><td>CORNING</td><td>30-002-CI</td><td>Component of cell culturing medium</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma Aldrich</td><td>F2442</td><td>Component of cell culturing medium</td></tr><tr><td>DMEM 1X</td><td>GIBCO</td><td>21068028</td><td>culturing medium for phoenix cell</td></tr><tr><td>CaCl2</td><td>Sigma Aldrich</td><td>C1016</td><td>used in phoenix cell transfection</td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H3375</td><td>used in phoenix cell transfection (HBS solution)</td></tr><tr><td>KCl</td><td>Sigma Aldrich</td><td>P9333</td><td>used in phoenix cell transfection (HBS solution)</td></tr><tr><td>Dextrose</td><td>Sigma Aldrich</td><td>D9434</td><td>used in phoenix cell transfection (HBS solution)</td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>S7653</td><td>used in phoenix cell transfection (HBS solution) and retrovirus precipitation</td></tr><tr><td>Na2HPO4</td><td>Sigma Aldrich</td><td>S3264</td><td>used in phoenix cell transfection (HBS solution)</td></tr><tr><td>TRYPSIN EDTA SOLUTION 1X</td><td>CORNING</td><td>59430C</td><td>used in cell split</td></tr><tr><td>DPBS 1X</td><td>GIBCO</td><td>14190250</td><td>Dulbecco&#039;s Phosphate Buffered Saline</td></tr><tr><td>DMSO</td><td>Sigma Aldrich</td><td>D8418</td><td>Component of cell freezing medium (80% FBB and 20% DMSO)</td></tr><tr><td>G-418 Disulphate</td><td>Formedium</td><td>G4185</td><td>selection drug for&amp;nbsp;</td></tr><tr><td>Gelatin solution Bioreagent</td><td>Sigma Aldrich</td><td>G1393</td><td>cotaing of 96 well DNA plate and freezing plate</td></tr><tr><td>Tris-base</td><td>Fisher BioReagents</td><td>10376743</td><td>Component of Cell lysis buffer for genomic DNA extraction</td></tr><tr><td>EDTA</td><td>Sigma Aldrich</td><td>E6758</td><td>Component of Cell lysis buffer for genomic DNA extraction</td></tr><tr><td>SDS</td><td>Sigma Aldrich</td><td>71729</td><td>Component of Cell lysis buffer for genomic DNA extraction</td></tr><tr><td>Proteinase K</td><td>Thermo Fisher</td><td>AM2546</td><td>Component of Cell lysis buffer for genomic DNA extraction</td></tr><tr><td>RNAse A</td><td>Thermo Fisher</td><td>12091021</td><td>RNA degradation during DNA extraction</td></tr><tr><td>Agarose</td><td>Sigma Aldrich</td><td>A5304</td><td>DNA gel preparation</td></tr><tr><td>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><td>ethanol</td><td>Fisher BioReagents</td><td>BP28184</td><td>DNA precipitation</td></tr><tr><td>Sodium Acetate&amp;nbsp;</td><td>Sigma Aldrich</td><td>71196</td><td>Used for DNA precipitation at a 3M concentration pH5.2</td></tr><tr><td>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><td>Trizol</td><td>AMBION</td><td>15596018</td><td>Organic solvent used for RNA extraction</td></tr><tr><td>Dnase I</td><td>THERMO SCIENTIFIC</td><td>89836</td><td>degradation of genomic DNA from RNA&amp;nbsp;</td></tr><tr><td>dNTPs mix</td><td>Invitrogen</td><td>10297018</td><td>used in RT and PCR reactions</td></tr><tr><td>DTT</td><td>Invitrogen</td><td>707265ML</td><td>used in RT reactions</td></tr><tr><td>diethyl pyrocarbonate</td><td>Sigma Aldrich</td><td>D5758</td><td>used to inactivate RNAses in water (1:1000 dilution)</td></tr><tr><td>Ribolock</td><td>Thermo Fisher</td><td>EO0381</td><td>RNase inhibitor</td></tr><tr><td>MOPS</td><td>Sigma Aldrich</td><td>M9381</td><td>preparation of RNA gel</td></tr><tr><td>Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td>preparation of denaturating RNA gel (1% PFA in 1x MOPS)</td></tr><tr><td>Superscript III Reverse transcriptase</td><td>Invitrogen</td><td>18080-093</td><td>Retrotranscription reaction</td></tr><tr><td>Pfu DNA polymerase (recombinant)</td><td>Thermo Scientific</td><td>EP0501</td><td>PCR reaction</td></tr><tr><td>2X qPCRBIO SyGreen Mix Separate-ROX</td><td>PCR BIOSYSTEMS</td><td>PB 20.14</td><td>qPCR reaction</td></tr><tr><td>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><td>Sodium Butyrate</td><td>Sigma Aldrich</td><td>B5887</td><td>used to promote retrovirus particles production in phoenix cells</td></tr><tr><td>PEG8000</td><td>Sigma Aldrich</td><td>89510</td><td>Precipitation of retrovirus partcles</td></tr><tr><td>35&amp;micro;-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 ...

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

Research

JoVE Journal

Genetics

7.3K Views.  University of Trento. Deze methode kan helpen bij het beantwoorden van belangrijke vragen over Telomeren, zoals gist telomeric noncoding RNA TERRA lokaliseren, en ik kan erop aandringen dat het telomeer van oorsprong of in trans, een andere cromes-mants Het belangrijkste voordeel van deze techniek is dat het onderzoekers in staat stelt om TERRA moleculen te visualiseren uitdrukken van een enkele telomeer in levende cellen. Demonstreren van de procedure zal Claudio Oss Pegorar en Nicole Bettin, twee afgestudeerde s...

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

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase activity. Although TERT is named as a reverse transcriptase, structural and phylogenetic analyses of TERT demonstrate that TERT is a member of right-handed polymerases, and relates to viral RNA-dependent RNA polymerases (RdRPs) as well as viral reverse transcriptase. We firstly identified RdRP activity of human TERT that generates complementary RNA stand to a template non-coding RNA and contributes to RNA silencing in cancer cells. To analyze this non-canonical enzymatic activity, we developed RdRP assay with recombinant TERT in 2009, thereafter established in vitro RdRP assay for endogenous TERT. In this manuscript, we describe the latter method. Briefly, TERT immune complexes are isolated from cells, and incubated with template RNA and rNTPs including radioactive rNTP for RdRP reaction. To eliminate single-stranded RNA, reaction products are treated with RNase I, and the final products are analyzed with polyacrylamide gel electrophoresis. Radiolabeled RdRP products can be detected by autoradiography after overnight exposure.

Human telomerase reverse transcriptase (TERT) synthesizes not only telomeric DNA but also double-stranded RNA through RNA-dependent RNA polymerase activity. Here, we describe a newly established assay to detect RNA-dependent RNA polymerase activity of endogenous TERT.

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase ...

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

Immunology and Infection

Droplet Digital TRAP (ddTRAP): Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

The telomere repeat amplification protocol (TRAP) is the most widely used assay to detect telomerase activity within a given a sample. The polymerase chain reaction (PCR)-based method allows for robust measurements of enzyme activity from most cell lysates. The gel-based TRAP with fluorescently labeled primers limits sample throughput, and the ability to detect differences in samples is restricted to two fold or greater changes in enzyme activity. The droplet digital TRAP, ddTRAP, is a highly sensitive approach that has been modified from the traditional TRAP assay, enabling the user to perform a robust analysis on 96 samples per run and obtain absolute quantification of the DNA (telomerase extension products) input within each PCR. Therefore, the newly developed ddTRAP assay overcomes the limitations of the traditional gel-based TRAP assay and provides a more efficient, accurate, and quantitative approach to measuring telomerase activity within laboratory and clinical settings.

Droplet Digital TRAP ddTRAP: Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

We successfully converted the standard telomere repeat amplification protocol (TRAP) assay to be employed in droplet digital polymerase chain reactions. This new assay, called ddTRAP, is more sensitive and quantitative, allowing for better detection and statistical analysis of telomerase activity within various human cells.

The telomere repeat amplification protocol (TRAP) is the most widely used assay to detect telomerase activity within a given a sample. The polymerase ...

Cancer Research

Examining Tumor Cell Dissemination from Lung Metastases

Tracking Tumor Cell Dissemination from Lung Metastases Using Photoconversion

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional process wherein cells from the primary tumor disseminate and seed metastases, tumor cells in existing metastases can also redisseminate and give rise to new lesions in tertiary sites in a process known as &#34;metastasis-from-metastases&#34; or &#34;metastasis-to-metastasis seeding.&#34; Metastasis-to-metastasis seeding may increase the metastatic burden and decrease the patient&#39;s quality of life and survival. Therefore, understanding the processes behind this phenomenon is crucial to refining treatment strategies for patients with metastatic cancer.
Little is known about metastasis-to-metastasis seeding, due in part to logistical and technological limitations. Studies on metastasis-to-metastasis seeding rely primarily on sequencing methods, which may not be practical for researchers studying the exact timing of metastasis-to-metastasis seeding events or what promotes or prevents them. This highlights the lack of methodologies that facilitate the study of metastasis-to-metastasis seeding. To address this, we have developed - and describe herein - a murine surgical protocol for the selective photoconversion of lung metastases, allowing specific marking and fate tracking of tumor cells redisseminating from the lung to tertiary sites. To our knowledge, this is the only method for studying tumor cell redissemination and metastasis-to-metastasis seeding from the lungs that does not require genomic analysis.

Author Spotlight: Decoding Metastasis-to-Metastasis Seeding Using a New In Vivo Technique for Tracking Breast Cancer Spread 

We present a method for studying tumor cell redissemination from lung metastases involving a surgical protocol for the selective photoconversion of lung metastases, followed by the identification of redisseminated tumor cells in tertiary organs.

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional ...

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay.
In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain.
Compared to the traditional TRAP assay that utilize 32P labeled radioactive dCTP&#39;s for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region.

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

Neuroscience

Utilizing Murine Inducible Telomerase Alleles in the Studies of Tissue Degeneration/Regeneration and Cancer

Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that cause tissue degeneration during ageing. Cancer, with incidence rates greatly increasing with age, is characterized by short telomere lengths and high telomerase activity. To study the roles of telomere dysfunction and telomerase reactivation in ageing and cancer, the protocol shows how to generate two murine inducible telomerase knock-in alleles 4-Hydroxytamoxifen (4-OHT)-inducible TERT-Estrogen Receptor (mTERT-ER) and Lox-Stopper-LoxTERT (LSL-mTERT). The protocol describes the procedures to induce telomere dysfunction and reactivate telomerase activity in mTERT-ER and LSL-mTERT mice in vivo. The representative data show that reactivation of telomerase activity can ameliorate the tissue degenerative phenotypes induced by telomere dysfunction. In order to determine the impact of telomerase reactivation on tumorigenesis, we generated prostate tumor model G4 PB-Cre4 PtenL/L p53L/L LSL-mTERTL/L and thymic T-cell lymphoma model G4 Atm-/- mTERTER/ER. The representative data show that telomerase reactivation in the backdrop of genomic instability induced by telomere dysfunction can greatly enhance tumorigenesis. The protocol also describes the procedures used to isolate neural stem cells (NSCs) from mTERT-ER and LSL-mTERT mice and reactivate telomerase activity in NSCs in vitro. The representative data show that reactivation of telomerase can enhance the self-renewal capability and neurogenesis in vitro. Finally, the protocol describes the procedures for performing telomere FISH (Fluorescence In Situ Hybridization) on both mouse FFPE (Formalin Fixed and Paraffin Embedded) brain tissues and metaphase chromosomes of cultured cells.

Telomere and telomerase play essential roles in ageing and tumorigenesis. The goal of this protocol is to show how to generate two murine inducible telomerase knock-in alleles and how to utilize them in the studies of tissue degeneration/regeneration and cancer.

Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that ...

Medicine

Proximity Ligation Assay to Study Oncogene-Derived Transcription-Replication Conflicts

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. Conflicts between the replication and transcription machinery, termed transcription-replication conflicts (TRCs), pose a considerable risk to genome stability, a critical factor in cancer development. While several factors regulating these collisions have been identified, pinpointing primary causes remains difficult due to limited tools for direct visualization and clear interpretation. In this study, we directly visualize TRCs using a proximity ligation assay (PLA), leveraging antibodies specific to PCNA and phosphorylated CTD of RNA polymerase II. This approach allows precise measurement of TRCs between replication and transcription processes mediated by RNA polymerase II. The method is further enhanced through DNA primers conjugated covalently to these antibodies, coupled with PCR amplification using fluorescent probes, providing a highly sensitive and specific means of detecting endogenous TRCs. Fluorescence microscopy enables the visualization of these conflicts, offering a powerful tool to study genome instability mechanisms associated with cancer. This technique addresses the gap in direct TRC visualization, allowing for a more comprehensive analysis and understanding of the underlying processes driving genome instability in cells.

Here, we present a sensitive and specific method to detect oncogene-induced transcription-replication conflicts (TRCs) using the proximity ligation assay (PLA). This approach leverages specific antibodies targeting PCNA and phospho-CTD RNAPII, enabling the assessment of TRC prevalence between RNA polymerase II transcription and DNA replication machinery.

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Biology

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

A major approach in the field of mammalian cell biology is the manipulation of the expression of genes of interest in selected cell lines, with the aim to reveal one or several of the gene's function(s) using transient/stable overexpression or knockdown of the gene of interest. Unfortunately, for various cell biological investigations this approach is unsuitable when manipulations of gene expression result in cell growth/proliferation defects or unwanted cell differentiation. Therefore, researchers have adapted the Tetracycline repressor protein (TetR), taken from the E. coli tetracycline resistance operon1, to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells2,3. In short, TetR has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system) (Figure 1). Given the inconvenience that the Tet-off system requires the continuous presence of tetracycline (which has a half-life of about 24 hr in tissue cell culture medium) the Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector systems for cDNA expression as used here.
Shortly after establishment of RNA interference (RNAi) for gene knockdown in mammalian cells4, vectors expressing short-hairpin RNAs (shRNAs) were described that function very similar to siRNAs5-11. However, these shRNA-mediated knockdown approaches have the same limitation as conventional knockout strategies, since stable depletion is not feasible when gene targets are essential for cellular survival. To overcome this limitation, van de Wetering et al.12 modified the shRNA expression vector pSUPER5 by inserting a TO in the promoter region, which enabled them to generate stable cell lines with tetracycline-inducible depletion of their target genes of interest.
Here, we describe a method to efficiently generate stable human Tet-on cell lines that reliably drive either inducible overexpression or depletion of the gene of interest. Using this method, we have successfully generated Tet-on cell lines which significantly facilitated the analysis of the MST/hMOB/NDR cascade in centrosome13,14 and apoptosis signaling15,16. In this report, we describe our vectors of choice, in addition to describing the two consecutive manipulation steps that are necessary to efficiently generate human Tet-on cell lines (Figure 2). Moreover, besides outlining a protocol for the generation of human Tet-on cell lines, we will discuss critical aspects regarding the technical procedures and the characterization of Tet-on cells.

Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

A rapid and simple way to generate human cell lines with inducible and reversible cDNA overexpression or shRNA-mediated knock-down of the gene of interest. This method enables researchers to reliably and highly reproducibly manipulate cell lines that are difficult to alter by transient transfection methods or conventional knockdown/knockout strategies.

A major approach in  the field of mammalian cell biology is the manipulation of the expression of  genes of interest in selected cell lines, with the aim ...

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by crossing the &#39;Tet-On&#39; system oTet-Cre mouse with a novel reverse tetracycline transactivator (rtTA) transgene linked to the Tert promoter, which we have demonstrated marks a novel population of adult brain stem cells. Here, administration of the tetracycline derivative doxycycline to mTert-rtTA::oTet-Cre mice will indelibly mark a population of cells that express a 4.4 kb fragment of the promoter region of the gene Tert. When combined the Rosa-mTmG reporter, mTert-rtTA::oTet-Cre::Rosa-mTmG mice will express membrane tdTomato (mTomato) until doxycycline treatment induces the replacement of mTomato expression with membrane EGFP (mGFP) in cells that also express Tert. Therefore, when these triple-transgenic lineage tracing mice receive doxycycline (the &#34;pulse&#34; period during which TERT expressing cells are marked), these cells will become indelibly marked mGFP+ cells, which can be tracked for any desirable amount of time after doxycycline removal (the &#34;chase&#34; period), even if Tert expression is subsequently lost. Brains are then perfusion-fixed and processed for immunofluorescence and other downstream applications in order to interpret changes to stem cell activation, proliferation, lineage commitment, migration to various brain niches, and differentiation to mature cell types. Using this system, any rtTA mouse can be mated to oTet-Cre and a Rosa reporter to conduct doxycycline-inducible &#34;pulse-chase&#34; lineage&#160;tracing experiments using markers of stem cells.

The ability to permanently mark stem cells and their progeny with a fluorophore using an inducible transgenic lineage tracing mouse line allows for spatial and temporal analysis of activation, proliferation, migration, and/or differentiation in vivo. Lineage tracing can reveal novel information about lineage commitment, response to intervention(s), and multipotency.

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by ...

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

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