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

1 . Selection of Neomycin Resistant Clones

1. Grow AGS cells in Ham's F-12K (Kaighn'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 µg per mL of medium) at 37 °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.
2. The following day, replace the culturing medium with medium containing neomycin at 0.7 µg/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 cells, on the following day the cells can be split from a single well to a 10 cm dish containing selective medium.
3. Keep the cells in selective medium for 7-10 days, changing medium every one or two days, until single clones are visible.
4. Picking of cell clones
   1. Prepare a 96 well plate containing 10 µL of 0.25% trypsin in each well.
      NOTE: Prepare two 96 well plates in case more than 96 clones are expected to be picked.
   2. 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.
   3. 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.
   4. Pick single colonies using a 10 µL pipette. Attach the tip containing 5 µL 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.
   5. Place the cells from the colony into a well of the 96 well plate containing 10 µL of 0.25% trypsin.
   6. Incubate 5 min at room temperature, then fill the well with 150 µL of selective medium (Ham's F-12K (Kaighn's) medium supplemented with 10% FBS, 2 mM L-glutamine, penicillin (0.5 units per mL of medium), streptomycin (0.2 µg per mL of medium) and containing neomycin at a concentration of 0.7 µg/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.
   7. 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's F-12K (Kaighn's) medium supplemented with 10% FBS, 2 mM L-glutamine, penicillin (0.5 units per mL of medium), streptomycin (0.2 µg per mL of medium) and containing neomycin at a concentration of 0.7 µg/mL at 37 °C and 5% CO₂, until reaching 90% confluence.
5. Splitting of clones
   1. Prepare three 96 well plates coated with gelatin by adding 100 µL 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.
   2. Once clones reach 90% confluence, aspirate medium from each well of the 96 well plate, wash with PBS, add 30 µL of 0.25% trypsin per well, and incubate for 5 min at 37 °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.
   3. Add 70 µL of selective medium per well, disrupt cell clumps by pipetting up and down inside the wells, then transfer 30 µL of the 100 µL to the gelatinized DNA plate prefilled with 120 µL of selective medium per well and 30 µL to the gelatinized freezing plates prefilled with 50 µL medium (without selection).
   4. Place the DNA plate in the incubator and allow the cells to grow at 37 °C and 5% CO₂ until 90% confluence.
   5. Add 80 µL 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 °C.
   6. Add 90 µL 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.

2. Screening of Neomycin Resistant Clones

1. DNA extraction from the 96 well DNA plate.
   1. Once the clones cultured in the DNA plate reach 90% confluence, wash 2 times with PBS, then lyse with 50 µL 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 °C overnight.
   2. Add 100 µL 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.
   3. Remove the Et-OH/NaCl solution by inverting the plate and wash 3 times with 200 µL of 70% ethanol per well.
   4. Add 25 µL of RNAse A solution in distilled water and incubate at 37 °C for 1 h.
2. Use 3 µL 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 °C for 20 s as denaturation step and then 34 cycles at 98 °C 10 s, 58°C 20 s, 72 °C 15 s, using polymerase enzyme in a 25 µL reaction mix (see Table of Materials). Primer sequences are indicated in Table 1.
3. 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).
4. 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.
5. Southern blot screening of PCR positive clones
   1. Grow the clones positive at PCR and sequencing screening from the original 96 well plate (the backup plate) to 6 well plates in Ham's F-12K (Kaighn's) medium supplemented with 10% FBS, 2 mM L-glutamine, penicillin (0.5 units per mL of medium), streptomycin (0.2 µg per mL of medium) and containing neomycin at a concentration of 0.7 µg/mL at 37 °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).
   2. Once the clones are at 90% confluence in the 6 well plate, wash the cells with PBS and add 250 µL of lysis buffer containing 0.5 µg proteinase K per well.
   3. Scrape the cells using a cell scraper and transfer the lysate in a 1.5 mL tube.
   4. Incubate at 37 °C for 16 h.
   5. Add 1 mL of 100% ethanol, shake vigorously, and allow the DNA to precipitate at least 2 h or overnight at -20 °C.
      NOTE: The protocol can be paused here.
   6. Spin at 13,400 x g at 4 °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.
   7. Resuspend the DNA pellets in 50 µL of distilled water containing RNAse A and incubate for 1 h at 37 °C.
   8. Digest 5-10 µg of genomic DNA using NcoI and BamHI restriction enzymes (two independent digestions) in 100 µL reaction volume by incubating the digestion reactions at 37 °C overnight.
   9. Run 4 µL of the digestion on agarose gel (0.8% agarose) for complete digestion confirmation.
   10. 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°C to precipitate DNA.
       NOTE: The protocol can be paused here.
   11. Centrifuge at 13,400 x g at 4 °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.
   12. Resuspend pellets in 20 µL 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 ( ̴30 volts). The electrophoresis set up should be optimized.
   13. The following day, stain the gel with a DNA labelling agent, such as ethidium bromide at 1 µL/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.
   14. Set the transfer of DNA to a nylon membrane and perform membrane hybridization with a MS2 sequence-specific probe using standard procedures²¹.
   15. Thaw the clones that are positive at PCR, DNA sequencing and Southern blot from one of the two freezing plates (see next step).
6. Thawing of clones
   1. Prepare one 15 mL tube containing 5 mL of pre-warmed Ham's F-12K (Kaighn's) medium supplemented with 10% FBS, 2 mM L-glutamine, penicillin (0.5 units per mL of medium), and streptomycin (0.2 µg per mL of medium) for each clone to be thawed.
   2. Remove one of the freezing plates from -80° and add 100 µL 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.
   3. 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.
   4. Aspirate the medium, resuspend the cells in 500 µL of pre-warmed complete F12K medium, and transfer each clone to a single well of a 12 well plate.
7. Elimination of the neomycin resistance gene from the MS2 cassette integrated at subtelomere 15q.
   1. 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.
   2. 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.
   3. 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).
   4. Culture the third dish containing the clone for cell freezing and RNA extraction.

3. Verification of TERRA-MS2 Transcript Expression by RT-qPCR

1. Upon verification of neomycin gene removal, perform total RNA extraction from TERRA-MS2 clones using organic solvents (phenol and guanidine isothiocynate solution)²¹.
   1. Resuspend the RNA extracted from a 10 cm dish in 100 µL of diethyl pyrocarbonate (DEPC) water.
   2. Run 3 µL 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.
   3. Treat 3 µg of RNA with DNAse I using 1 unit of DNAse I enzyme in 60 µL final reaction volume.
   4. Incubate the reaction for 1 h at 37 °C.
2. Reverse transcription reaction and qPCR analyses
   1. Add the following components to a nuclease-free microcentrifuge tube: 2 µL of a 1 µM TERRA specific primer, 1 µL of dNTPs mix (10 mM each), 6 µL of DNAse I-treated RNA (corresponding to ̴300ng RNA). Adjust the volume to 13 µL with 4 µL 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.
   2. Heat the mixture to 65 °C for 5 min and incubate in ice for at least 1 min.
   3. Collect the content of the tubes by brief centrifugation and add 4 µL of 5X RT enzyme Buffer, 1 µL 0.1 M dithiothreitol (DTT), 1 µL (4 units) of RNAse inhibitor, and 1 µL of reverse transcriptase (see Table of Materials).
   4. Incubate the samples at 42 °C for 60 min, then use 2 µL of the RT reaction for qPCR analyses.
3. Prepare qPCR reaction mix in a final volume of 20 µL consisting of 10 µL of 2x qPCR master mix, 2 µL of cDNA template, 1 µL of forward primer (10 µM), 1 µL of reverse primer (10 µM), and 6 µL of water.
4. Perform qPCR reaction in a thermocycler using standard protocols²¹.

4. Production of a MS2-GFP Expressing Retrovirus

1. 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.
2. 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.
3. Incubate for 8 h at 37 °C, 5% CO₂, then replace the medium with fresh culturing medium from which the virus will be collected.
4. 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 µm filter, add polybrene (30 µg/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 1/5^th volume of 50% PEG-8000/900 mM NaCl solution and incubating overnight at 4 °C on a rotator wheel.
5. 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 1/100^th 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.

5. Visualization of TERRA-MS2 Transcripts in Living Cells

1. Plate TERRA-MS2 clones and WT AGS cells in glass-bottomed dishes. On the day of infection, add polybrene to the medium (30 µg/mL final concentration) and the MS2-GFP expressing retrovirus.
2. After 24 hours, discard the virus-containing medium and add fresh medium without phenol-red.
3. 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 °C and 5% CO₂ during imaging.