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