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., 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 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 ...

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

Research

JoVE Journal

Genetics

7.3K Views.  University of Trento. 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.

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

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

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

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play in the functioning of cells and whole organisms. This cause has been tremendously aided by the recent introduction of the CRISPR/Cas9 system-a versatile tool that allows researchers to manipulate the genome and transcriptome in order to, among other things, knock out, knock down, or knock in genes in a targeted manner. For the purpose of knocking out a gene, CRISPR/Cas9-mediated double-strand breaks recruit the non-homologous end-joining DNA repair pathway to introduce the frameshift-causing insertion or deletion of nucleotides at the break site. However, an individual guide RNA may cause undesirable off-target effects, and to rule these out, the use of multiple guide RNAs is necessary. This multiplicity of targets also means that a high-volume screening of clones is required, which in turn begs the use of an efficient high-throughput technique to genotype the knockout clones. Current genotyping techniques either suffer from inherent limitations or incur high cost, hence rendering them unsuitable for high-throughput purposes. Here, we detail the protocol for using fluorescent PCR, which uses genomic DNA from crude cell lysate as a template, and then resolving the PCR fragments via capillary gel electrophoresis. This technique is accurate enough to differentiate one base-pair difference between fragments and hence is adequate in indicating the presence or absence of a frameshift in the coding sequence of the targeted gene. This precise knowledge effectively precludes the need for a confirmatory sequencing step and allows users to save time and cost in the process. Moreover, this technique has proven to be versatile in genotyping various mammalian cells of various tissue origins targeted by guide RNAs against numerous genes, as shown here and elsewhere.

The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

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 ...

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.&#160;

We show a method for necropsy and dissection of mouse prostate cancer models, focusing on prostate tumor dissection. A step-by-step protocol for generation of mouse prostate tumor organoids is also presented.

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

RNA Next-Generation Sequencing and a Bioinformatics Pipeline to Identify Expressed LINE-1s at the Locus-Specific Level

Long INterspersed Elements-1 (LINEs/L1s) are repetitive elements that can copy and randomly insert in the genome resulting in genomic instability and mutagenesis. Understanding the expression patterns of L1 loci at the individual level will lend to the understanding of the biology of this mutagenic element. This autonomous element makes up a significant portion of the human genome with over 500,000 copies, though 99% are truncated and defective. However, their abundance and dominant number of defective copies make it challenging to identify authentically expressed L1s from L1-related sequences expressed as part of other genes. It is also challenging to identify which specific L1 locus is expressed due to the repetitive nature of the elements. Overcoming these challenges, we present an RNA-Seq bioinformatic approach to identify L1 expression at the locus specific level. In summary, we collect cytoplasmic RNA, select for polyadenylated transcripts, and utilize strand-specific RNA-Seq analyses to uniquely map reads to L1 loci in the human reference genome. We visually curate each L1 locus with uniquely mapped reads to confirm transcription from its own promoter and adjust mapped transcript reads to account for mappability of each individual L1 locus. This approach was applied to a prostate tumor cell line, DU145, to demonstrate the ability of this protocol to detect expression from a small number of the full-length L1 elements.

Here, we present a bioinformatic approach and analyses to identify LINE-1 expression at the locus specific level.

Long INterspersed Elements-1 (LINEs/L1s) are repetitive elements that can copy and randomly insert in the genome resulting in genomic instability and ...