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Centromere Chromosome Orientation Fluorescent in situ Hybridization (Cen-CO-FISH) Detects Sister Chromatid Exchange at the Centromere in Human Cells
着丝粒染色体定位荧光原位杂交法(Cen-CO-FISH)检测人细胞中着丝粒DNA重复的姐妹染色单体交换事件   

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Proceedings of the National Academy of Sciences of the United States of America
Feb 2017

Abstract

Human centromeres are composed of large tandem arrays of repetitive alpha satellite DNA, which are often sites of aberrant rearrangement in cancers (Mitelman et al., 1997; Padilla-Nash et al., 2001). To date, annotation of the human centromere repetitive sequences remains incomplete, greatly hindering in-depth functional studies of these regions essential for chromosome segregation. In order to monitor sister chromatid exchange happening at the centromere (C-SCE) due to recombination and mutagenic events, I have applied the Chromosome-Orientation Fluorescence in situ Hybridization (CO-FISH) technique to centromeres (Cen-CO-FISH) in human cells. This hybridization-based method involves (1) the incorporation of nucleotide analogs through a single round of replication, (2) enzymatic digestion of the newly synthesized DNA strand and (3) subsequent hybridization of single-stranded probes, in absence of a denaturation step. The resulting signal allows to differentially label each sister chromatid based on the 5’-3’ directionality of the DNA and to score aberrant staining patterns indicative of C-SCE. The Cen-CO-FISH method applied to human centromeres revealed that human centromeres indeed undergo recombination in cycling cells resulting in C-SCE, and centromere instability is enhanced in cancer cell lines and primary cells undergoing senescence (Giunta and Funabiki, 2017). Here, I present the detailed protocol of the preparation, experimental procedure and data acquisition for the Cen-CO-FISH method in human cells. It also includes a conceptual overview of the technique, with examples of representative images and scoring guidelines. The Cen-CO-FISH represents a valuable tool to facilitate exploration of centromere repeats.

Keywords: Centromere (着丝粒), Fluorescence in situ hybridization (荧光原位杂交), CO-FISH (CO-FISH), Alpha satellite (α卫星), Repetitive DNA (重复DNA), Genome stability (基因组稳定性), Recombination (重组), Sister chromatid exchange (姐妹染色单体交换)

Background

The human genome project was marked completed in 2003, yet it omitted over 10% of the human repetitive DNA (de Koning et al., 2011), including the centromere. The human centromere is a highly specialized genomic locus (Choo, 1997) playing a critical role during chromosome segregation where it serves as the site of kinetochore assembly to allow interaction with microtubules and sister chromatids separation during cell division (Cheeseman, 2014). Human centromeres are made of characteristic repetitive DNA sequences called alpha-satellites, whose linear assembly remains largely absent from the reference genomes. Here, I present the application of the Cen-CO-FISH technique to label human centromere and monitor recombination events resulting in crossover. Introduced by Bailey and colleagues over 20 years ago (Bailey et al., 1996), the CO-FISH method has been widely applied to detect recombination, fragility, replication timing, fusion and inversions at telomeres repeats, as well as to monitor mitotic segregation patterns and non-random sister chromatid segregation (Bailey et al., 2010). The application of this methodology to centromere, hereby called Cen-CO-FISH method, has revealed that the centromere-specific histone variant CENP-A, and CENP-A associated proteins CENP-C and CENP-T/W, work to prevent centromere instability and this functionality is compromised in cancer cell lines and in primary cells approaching replicative senescence that display higher number of C-SCE (Giunta and Funabiki, 2017). Cen-CO-FISH was used to assess centromere instability in cancer and during cellular senescence in human cells (Giunta and Funabiki, 2017) and it has been previously applied to study recombination (Jaco et al., 2008; de La Fuente et al., 2015) and sister chromatid separation patterns in mouse cells (Falconer et al., 2010). The wide application potentials of this methodology spans from quantitative detection of alpha satellite repeats, centromere recombination resulting in C-SCE, fragility, replication timing, fusion and inversions, as well as to monitor mitotic segregation patterns and non-random sister chromatids segregation (Bailey et al., 2010; Yadlapalli and Yamashita, 2013). Cen-CO-FISH fills the gaps in the missing genetic information that have cast a shadow over the centromere and other repetitive regions, bringing new light into the possibilities for functional exploration of these important loci of our genome.

Materials and Reagents

  1. 6 or 10 cm Petri dish (Corning, Falcon®, catalog numbers: 353002 or 353003 )
  2. 15 ml Falcon tube (Corning, Falcon®, catalog number: 352097 )
  3. Frosted slides (Superfrost Plus; Fisher Scientific, catalog number: 12-550-15 )
  4. Coverslips (24 x 60 mm) (Fisher Scientific, catalog number: 12-545-M )
  5. Paper towel
  6. Glass Pasteur pipette (Fisher Scientific, catalog number: 13-678-20A )
  7. Gloves and lab coat
  8. Human cells of interest and appropriate medium
    Note: This protocol is for adherent cells, changes can be made for use for non-adherent cultures.
  9. 5’-Bromodeoxyuridine (BrdU) (MP Biomedicals, catalog number: 100166 )
    Note: Prepare 10 mM stock solution in double distilled water (1,000x); make aliquots and store at -20 °C.
  10. 5’-Bromodeoxycytidine (BrdC) (Sigma-Aldrich, catalog number: B5002 )
    Note: Prepare 10 mM stock solution in double distilled water (1,000x); make aliquots and store at -20 °C.
  11. Colcemid (Roche Diagnostics, catalog number: 10295892001 ; already diluted 10 μg/ml)–Store at 4 °C
  12. Phosphate-buffered saline (PBS)
  13. Trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25300 )
  14. Fetal bovine serum (Atlanta Biologicals)
  15. Potassium chloride (KCl) (Fisher scientific, catalog number: P217-500 )
  16. RNase A (Sigma-Aldrich, catalog number: R5000 )
    Note: Prepare stock solution 50 mg/ml in 10 mM Tris-HCl pH 7.2. Aliquot and store at -20 °C.
  17. Hoechst 33258 (Thermo Fisher Scientific, InvitrogenTM, catalog number: H3569 )
    Note: Make a 10 μg/ml solution in double distilled water and store at 4 °C away from light.
  18. Exonuclease III and buffer (Promega, catalog number: M1811 )–Keep at -20 °C
  19. DAPI (Sigma-Aldrich, catalog number: D9542 )–0.5 mg/ml stock in water. Keep at 4 °C in the dark for one year
  20. ProLong Gold Anti-fade Reagent (Thermo Fisher Scientific, InvitrogenTM, catalog number: P36934 )
  21. Nail varnish (Sally Hansen, Transparent Harderer)
  22. Methanol (Fisher Scientific, catalog number: A452-4 )
  23. Glacial acetic acid (Fisher Scientific, catalog number: A38C-212 )
  24. Ethanol 100% (Decon, catalog number: 2716 ), 90% and 70%
  25. Blocking reagent (Roche Diagnostics, catalog number: 11096176001 )
  26. Maleic acid (Sigma-Aldrich, catalog number: M0375 )
  27. Sodium chloride (NaCl) (Merck, catalog number: SX0420-5 )
  28. Sodium hydroxide (NaOH) (Fisher Scientific, catalog number: S318-500 )
  29. Tris-HCl pH 7.2 (Sigma-Aldrich, CAS number: 1185-53-1)
  30. Formamide (Fisher Scientific, catalog number: BP228 ; use deionized for hybridization)
  31. Bovine serum albumin (BSA)
  32. Tween-20 (Hoefer, CAS number: GR128-500)
  33. Sodium citrate (Sigma-Aldrich, CAS number: 6132-04-3)
  34. Magnesium chloride (MgCl2) (Fisher Scientific, catalog number: AC41341-5000)
    Manufacturer: Acros Organics, catalog number: 413410025 .
  35. Dithiothreitol (DTT) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0861 )
  36. Hypotonic solution (see Recipes)
  37. Fixative solution (see Recipes)
  38. Ethanol dilution (see Recipes)
  39. Blocking solution (see Recipes)
  40. Hybridization solution (see Recipes)
  41. Hybridization wash #1 (see Recipes)
  42. Hybridization wash #2 (see Recipes)
  43. Peptide nucleic acid (PNA) probes (custom probes from PNABio) (see Recipes)
  44. Sodium chloride and sodium citrate buffer (SSC, see Recipes)

Equipment

  1. Pipettes (Gilson)
  2. Centrifuge (Eppendorf, model: 5810 R )
  3. Coplin Jars (Scienceware, Sigma-Aldrich, catalog number: S5641 )
  4. Heating block (VWR)
  5. Water bath (Fisher Scientific, model: IsotempTM 210 )
  6. Stratalinker with 365-nm UV light blubs (Spectralinker XL-1000 1800 UV irradiator) (Spectronics Corporation, model: XL-1000 )
  7. Slides plastic tray–to fit into the Stratalinker
  8. Hybridization chamber (see text for more details)
  9. Orbital shaker
  10. Imaging equipment:
    1. DeltaVision Image Restoration microscope system (Applied Precision/GE Healthcare)
    2. Olympus IX-70 microscope (Olympus, model: IX70 )
    3. 100x/1.40 UPLSAPO objective lens
    4. CoolSnap QE CCD camera (Photometrics)

Software

  1. SoftWoRx (Sold by Applied Precision)
  2. Metamorph 7.8 (Sold by Universal Imaging)
  3. Prism 5 (Sold by GraphPad)

Procedure

  1. Incorporation of BrdU:C and metaphase chromosome spread preparation
    1. Cells are seeded into a 6 or 10 cm dish at least two days before harvesting, to be about 70% confluent at the time of harvesting. In a 10 cm dish, seed ~1 million cells.
    2. Cells are then labeled for 16-20 h by incubating with 5’-Bromodeoxyuridine (BrdU) and 5’-Bromodeoxycytidine (BrdC). Thaw a 1,000x aliquot of BrdU and BrdC and mix 3:1 to obtain 7.5 mM BrdU + 2.5 mM BrdC. Add to the cells medium at a 1:1,000 dilution and incubate the cells overnight.
      Note: Adjust the time of BrdU:C labeling according to whether your cells grow slower/faster to avoid double labeling or incompletely labeled cells. You are aiming for cells to incorporate BrdU and BrdC throughout S-phase and to prevent them to exit from mitosis after replication. Typically, HeLa cells are incubated with BrdU:C for 14-16 h before adding colcemid; to validate the time of BrdU:C incubation one can synchronize cells with thymidine at G1/S, release and monitor exit from mitosis by flow cytometry. Cells that exit mitosis 15 h post-thymidine release should be treated with BrdU:C for 16-17 h before addition of colcemid to ensure only cells that started labeling before S-phase are trapped by the colcemid block.
    3. Add colcemid to a final concentration of 0.1 µg/ml (1:100 dilution of 10 µg/ml stock) to accumulate mitotic cells for about 2 h before harvesting.
      Note: Avoid leaving cells into colcemid for over 3 h, because long exposure to colcemid will lead to very compacted chromosome morphology.
    4. Harvest floating cells by saving the supernatant in a 15 ml Falcon tube, wash the dish once with PBS and collect the PBS wash as well into the same tube. Trypsinize for 5 min, block with medium containing fetal bovine serum, and collect the rest of the cells in the same tube.
    5. Pellet cells for 5 min at 175 x g. Remove supernatant and resuspend cells in 10 ml of pre-warmed 75 mM KCl solution. Gently pipette up and down to resuspend the pellet.
    6. Incubate the cells in the KCl hypotonic solution at 37 °C for 30 min (slowly invert the tube every 10 min to keep the cells suspended). Before pelleting the cells, adding 200 µl of fixative solution and mixing by slowly inverting the tubes twice helps the metaphases to remain intact during centrifugation, because cells are extremely fragile after the hypotonic treatment. Pellet the cells for 5 min at 175 x g.
    7. Aspirate most supernatant, leaving about 200 µl of KCl to resuspend cells by tapping the tube or slowly pipetting up and down with a P1000.
    8. Fix the resuspended pellet by adding 10 ml of freshly made ice-cold MeOH-acetic acid (3:1) fixative solution dropwise (prepare it 30 min before use, keep at -20 °C) while continuously vortexing the tube on a Vortex mixer at the lowest speed setting (#1).
      Note: It is important to resuspend the pellet before addition of the fixative, and to add the fixative one drop at a time while continuously vortexing the cells at low speed for at least the first 2 ml. The remaining 8 ml can be added all at once.
    9. Mix by inverting the tube a few times.
    10. Cells can now be kept at 4 °C for several months, stored away from light.
    11. Once you are ready to drop the cells, spin the tube down at 380 x g for 5 min. Aspirate the fixative leaving about 0.5-2 ml depending on how many cells are in each tube. Thoroughly resuspend cells in the remaining fixative.
    12. If you are dropping onto Superfrost Plus slides, go directly to Step A13. If using regular slides, wash in soapy water and rinse 3 times with Milli-Q water before use. Keep slides in Milli-Q water in the fridge for an hour and dry well right before use.
    13. Humidify the slide you are about to ‘drop’ on by hovering it over a hot wet towel placed on a heating block, until it steams up. You can also achieve this by ‘breathing’ onto the slide.
      Note: Creating conditions of humidity, for instance by having wet paper towels onto an adjacent 90 °C block that continues to release vapor, and humidifying the air immediately adjacent to where slides are dropped, or using a hybridization hood with set humidity and temperature, helps with the quality of the spreads.
    14. Using a glass Pasteur pipette, pipette up and down and aspirate the cell suspension. Drop several drops of cell suspension onto different places on the slide from about 20 cm away. You should aim to cover all parts of the slide with each drop.
      Note on safety: Gloves and lab coat should be used to protect from possible splashes. The entire metaphase spread procedure can be performed in a laboratory chemical fume hood for additional protection.
    15. Immediately after dropping, rest the slide for about 2-3 min onto a 42 °C block with a wet paper towel covering the block, cells side up.
      Note: Factors that influence the spreading are: humidity, temperature, distance of dropping cells onto the slide and drying time.
    16. Air-dry slides overnight at room temperature away from light. You can keep any cells left into the falcon tube stored in fresh fixative at 4 °C for up to one year.

  2. Enzymatic digestion of BrdU:C labeled, newly synthesized strand
    1. Dropped slides are inserted in a Coplin jar and rehydrated in PBS (pH 7.0-7.5) for 5 min (about 35 ml of solution is needed for each jar).
    2. Treat slides with 0.5 mg/ml RNase A (in PBS, DNase free) for 10 min in a 37 °C water bath.
    3. Stain slides with 1 µg/ml Hoechst 33258 (Sigma-Aldrich) diluted into 2x SSC for 15 min at room temperature with slight agitation.
    4. Place slides in a shallow plastic tray and add just enough 2x SSC buffer to cover the slides. Expose the slides to 365 nm UV light at room temperature for 1,800 sec (30 min; equivalent to 5,400 J/m2) in a Stratalinker 1800.
    5. Digest the BrdU:C labeled UV nicked DNA strand with 80 µl of 10 U/µl Exonuclease III (Promega) into buffer supplied by the manufacturer diluted to 1x (50 mM Tris-HCl, 5 mM MgCl2, 5 mM DTT, pH 8.0). Add the 80 µl solution onto a 24 x 60 coverslip that will make contact with the entire surface of the cells on the slide. Let it adhere to the slide, then place the slide cells side up for 10 min, away from light. Avoid air bubbles.
    6. Remove the coverslips and repeat the Exonuclease III digestion step with fresh solution for another 10 min.
    7. Wash slides in PBS for 5 min and dehydrate successively in 70%, 90%, 100% ethanol series for 2 min each.
    8. Air dry slides and store at room temperature in the dark (can be left overnight).

  3. Fluorescent in situ hybridization
    1. Place 80 µl of hybridization mix onto coverslips and pick up the coverslip with the slide, then put the slide into the hybridization chamber (see Figures 1G-1I for instructions on how to make a hybridization chamber and for a visual illustration of these steps). Avoid air bubbles.


      Figure 1. Representative images of the Cen-CO-FISH procedure at different stages of the protocol. A. Cells are harvested, hypotonically swollen and fixed overnight. Samples can be stored at 4 °C at this stage. Cells are then dropped onto glass slides to let the metaphases spread. Slides can be stored in the dark at room temperature (RT) before proceeding to Step B. B. Rehydrate the slides into PBS in a Coplin jar for 5 min. C. Remove PBS and add RNase A solution into the jar, incubate in a 37 °C water bath for 10 min. Remove RNase solution and incubate with Hoechst in 2x SSC for 15 min at RT. D. Place slides into plastic try and cover with just enough 2x SSC. E. Place tray into Stratalinker oven with 365 nm UV bulbs and expose for 30 min (1,800 sec). F. Prepare 80 μl of Exonuclease III solution for each slide and add the solution to a 24 x 60 coverslip that will make contact with the entire surface of the slide. G. Immediately after UV exposure, pick up the coverslip with Exonuclease III solution as shown. Invert the slide, adjust the coverslip to make sure it is central, the liquid is well distributed and there are no air bubbles. Incubate cells side up for 10 min at RT and then repeat Steps F-G one more time for an additional Exonuclease incubation. Wash in PBS and dehydrate in ethanol series. Store slides at RT in the dark (overnight). H. Prepare one of the probes (1:1,000-1:5,000) into hybridization solution, heat for 10 min at 60 °C before adding 80 μl onto the coverslips. Proceed as in Steps F-G shown. I. Place slides into a Hybridization chamber and incubate for 2 h at RT. To make a chamber, take a box, fill it with paper towels and wet throughout until all towels are humid. Do not put the slides directly on top of the wet paper. Use pipettes or other forms of support to raise the coverslips over the wet paper. Place slides cells side up and close the box away from light. Upon opening the box after 2 h, you should see a small amount of condensation on the lid. Wash with Hybridization buffer #1 and repeat Step H with the second, reverse complement probe (you can invert the order of the hybridization between forward and reverse complement probes, it should yield identical results). Wash in Hybridization buffer #1 and #2, including the DAPI step, dehydrate and air dry for at least one hour. Mount and seal before imaging.

    2. Hybridize with the Forward PNA probe at a 1:1,000 dilution (heated at 60 °C for 10 min right before use) in the dark for 2 h in the hybridization chamber.
    3. Rinse in Wash #1 for 30 sec. Leave slides draining vertically for 10 sec.
    4. Hybridize with the Reverse Complement PNA probe at a 1:1,000 dilution (heated at 60 °C for 10 min right before use) in the dark for 2 h.
      Note: Inverting the order of the probes will not affect the hybridization or quality of the signal.
    5. Wash slides in Wash #1 for 15 min 2 times on an orbital shaker.
    6. Wash slides in Wash #2 for 5 min 3 times. To the second wash, add 1:500 DAPI from 0.5 mg/ml stock.
    7. Dehydrate in 70%, 90%, 100% ethanol series for 3 min each.
    8. Air dry slides for about 1 h.
    9. Mount slides using Prolong Gold embedding medium (avoid bubbles) and seal using nail varnish. Slides are ready for imaging.
    10. Slides can be stored up to 1 week at 4 °C or at -20 °C for longer storage. 

Data analysis

Image acquisition and processing

  1. Cells are imaged using a DeltaVision Image Restoration microscope system (Applied Precision/GE Healthcare), mounted on an Olympus IX-70 microscope and fitted with a 100x/1.40 UPLSAPO objective lens and a CoolSnap QE CCD camera (Photometrics).
  2. 15 or more metaphase spreads for each experiment are imaged to yield statistically significant and representative results. When imaging the cells, care should be taken to select the best spreads where chromosomes are nicely separated (Figure 2A) and approximately 46 chromosomes are present in the case of karyotypically stable diploid cells. Typical exposure times for the DAPI is 0.3 sec, for the TRITC channel (red probe) is 0.1 sec and for the FITC (green probe) is 0.5 sec.
  3. The image is acquired as a z-stack containing about 15-30 0.2 μm sections. Acquired images are deconvolved using SoftWoRx (Applied Precision) and exported to Metamorph (Universal Imaging) for analysis as maximum projections, as shown in the examples in Figures 2A and 2B.

Representative images and scoring

  1. Following the Cen-CO-FISH protocol outlined, high-quality metaphase spreads are obtained stained with a specific centromere marker, as shown in Figure 2A. The forward probe, labeled in red, and the reverse strand probe, labeled in green, should not overlap but be adjacent to each other, labeling one individual sister chromatid of each chromosome (Figure 2B). Quantifications are performed by counting the number of aberrant Cen-CO-FISH patterns indicative of C-SCE in each metaphase spreads (Figure 2A, colored boxes). Examples of normal (top panel, teal box; Figure 2B) and aberrant Cen-CO-FISH pattern indicative of C-SCE are shown (bottom panels, red boxes; Figure 2B). In high chromosomal instability (CIN) lines or some cancer cell lines, a small number of aberrant patterns can be found resulting from fusion events occurring outside of the centromere region, as indicated by the presence of DAPI separating the two pairs of centromere signal, and should be excluded from the quantification of C-SCE (grey box; Figure 2C). The number of C-SCE can be presented as the percentage of aberrant centromeres over the total number of labeled centromere pairs for each metaphase and plotted in a scatter plot using Prism, as previously shown (Giunta and Funabiki, 2017).


    Figure 2. Detecting recombination events at human centromere using the Cen-CO-FISH method. A. HeLa cells were stained with Cen-CO-FISH using forward (red) and reverse (green) probes raised against the CENP-B box sequence, present in all chromosomes in the metaphase spread shown in A. Individual channels allow the visual identification of aberrant Cen-CO-FISH staining patterns, often present as a ‘double dot’ for the red channel and the same for the green channel, as indicated by the white arrows. Color combined image merged with DAPI is also shown. Boxes are enlarged in Figure 2B. Scale bar = 5 µm. B. Enlarged boxes from Figure 2A, left panel, showing a representative image for a normal Cen-CO-FISH staining pattern (marked in teal), and three panels below showing aberrant patterns (marked in red) indicative of C-SCE. Scale bar = 1 µm. C. Examples of an aberrant staining pattern resulting from recombination outside the centromere sequence (marked in grey), thus excluded from quantification of centromere recombination and C-SCE. Scale bar = 1 µm.

  2. Cen-CO-FISH is a reliable, reproducible and sensitive labeling technique to visualize repetitive genomic regions. The technique relies on two crucial steps: repetitiveness of the locus and incorporation of BrdU and BrdC into the newly synthesized strand. The probes used for the Cen-CO-FISH hybridization are fluorescently-labeled PNA 17 or 18-mer. Designing the probes to be aimed at sequences that are repeated enables the amplification of the fluorescence intensity. Centromere alpha satellite repeats spans from 0.5-5 Mb with sequences common to each human chromosomes (Schueler et al., 2001; Fukagawa, 2004), yielding a robust Cen-CO-FISH signal for visualization, acquisition using conventional microscopy, and functional analyses. The reliance of the technique on labeling of the newly synthesized strand, which will subsequently be enzymatically digested, signify that this method can only be applied to cycling cells. However, this also represents an advantage for this method, where the yielded patterns are indicative of recombination events having happened during a single prior cell cycle. Thus, Cen-CO-FISH provides a bird’s-eye view on these dark genomic regions while giving specific temporal resolution and sequence orientation information to examine the behavior of the centromere repeats. Previous studies have reported the application of the Cen-CO-FISH technique to monitor centromere recombination in mouse cells (Bailey et al., 1996; Jaco et al., 2008; Falconer et al., 2010) indicating that the technique can be successfully applied to tissue culture cells from different species using specifically designed probes.

Recipes

  1. Hypotonic solution
    75 mM KCl
    Prewarm the required amount in a 37 °C water bath before each use
  2. Fixative solution
    3 parts methanol
    1 part glacial acetic acid
    Must be made fresh each time
    Use ice-cold
  3. Ethanol dilution
    70% and 90% in double distilled water
  4. Blocking solution
    Dissolve the blocking reagent (Roche) into maleic acid buffer to make a 10% stock:
    100 mM maleic acid
    150 mM NaCl
    Adjust pH to 7.5 with NaOH and store at 4 °C
    Shake vigorously before each use
  5. Hybridization solution
    10 mM Tris-HCl pH 7.2
    70% formamide
    0.5% blocking reagent (from 10% stock)
  6. Hybridization wash #1
    10 mM Tris-HCl pH 7.2
    70% formamide (Sigma-Aldrich)
    0.1% BSA (dissolve in double distilled water before adding formamide)
  7. Hybridization wash #2
    0.1 M Tris-HCl pH 7.2
    0.15 M NaCl
    0.1% Tween-20
    For the DAPI wash, add 1:500 DAPI using the 0.5 mg/ml stock
  8. Peptide nucleic acid (PNA) probes (custom probes from PNABio)
    Set #1 against the alpha satellite:
    F3003 CENT-Cy3 (Cy3–OO–AAACTAGACAGAAGCATT)
    Reverse complement CENT-RC-488 (Alexa488–O–AATGCTTCTGTCTAGTTT)
    Set #2 against the CENP-B box:
    F3002 CENP-B Cy3 (ATTCGTTGGAAACGGGA)
    Reverse complement CENP-B-RC-488 (TCCCGTTTCCAACGAAT)
    Notes:
    1. Lyophilized probes were diluted to 50 μM in double distilled water based on manufacturer’s instructions. Aliquot and store at -80 °C to avoid multiple freeze-thawing.
    2. For hybridization, thaw and spin the probes, and dilute into hybridization solution at a 1:1,000-1:5,000 dilution (adjust according to the signal obtained). Heat at 60 °C for 10 min before use.
  9. Sodium chloride and sodium citrate buffer (SSC; 20x)
    NaCl (3 M), sodium citrate (300 mM) dissolved in distilled water
    Adjust pH to 7.0 and autoclave
    Working dilution is 2x

Acknowledgments

I would like to thank H. Funabiki, F. Lottersberger, N. Bosco, and T. DeLange, as well as to K. Thomas, A. North and T. Tong at the Rockefeller University Bio-Imaging Resource Center (BIRC). I also want to thank Seneca Jason and A. Field for their support throughout this work. S.G. was funded by American-Italian Cancer Foundation fellowship and Women in Science Rockefeller fellowship. The Laboratory of Chromosome and Cell Biology where the work has been carried out is funded by a grant to the Head of Laboratory Prof. Hironori Funabiki from the National Institute of Health (NIH) (R01GM075249). This protocol was adapted from procedures published in Giunta and Funabiki (2017) and Celli et al. (2006).
Competing interests statement: The author declares no competing interests.

References

  1. Bailey, S. M., Goodwin, E. H., Meyne, J. and Cornforth, M. N. (1996). CO-FISH reveals inversions associated with isochromosome formation. Mutagenesis 11(2): 139-144.
  2. Bailey, S. M., Williams, E. S., Cornforth, M. N. and Goodwin, E. H. (2010). Chromosome Orientation fluorescence in situ hybridization or strand-specific FISH. Methods Mol Biol 659: 173-183.
  3. Celli, G. B., Denchi, E. L. and de Lange, T. (2006). Ku70 stimulates fusion of dysfunctional telomeres yet protects chromosome ends from homologous recombination. Nat Cell Biol 8(8): 885-890.
  4. Cheeseman, I. M. (2014). The kinetochore. Cold Spring Harb Perspect Biol 6(7): a015826.
  5. Choo, K. H. A. (1997). The centromere. Oxford University Press, USA.
  6. de Koning, A. P., Gu, W., Castoe, T. A., Batzer, M. A. and Pollock, D. D. (2011). Repetitive elements may comprise over two-thirds of the human genome. PLoS Genet 7(12): e1002384.
  7. de La Fuente, R., Baumann, C. and Viveiros, M. M. (2015). ATRX contributes to epigenetic asymmetry and silencing of major satellite transcripts in the maternal genome of the mouse embryo. Development 142(10): 1806-1817.
  8. Falconer, E., Chavez, E. A., Henderson, A., Poon, S. S., McKinney, S., Brown, L., Huntsman, D. G. and Lansdorp, P. M. (2010). Identification of sister chromatids by DNA template strand sequences. Nature 463(7277): 93-97.
  9. Fukagawa, T. (2004). Centromere DNA, proteins and kinetochore assembly in vertebrate cells. Chromosome Res 12(6): 557-567.
  10. Giunta, S. and Funabiki, H. (2017). Integrity of the human centromere DNA repeats is protected by CENP-A, CENP-C, and CENP-T. Proc Natl Acad Sci U S A 114(8): 1928-1933.
  11. Jaco, I., Canela, A., Vera, E. and Blasco, M. A. (2008). Centromere mitotic recombination in mammalian cells. J Cell Biol 181(6): 885-892.
  12. Mitelman, F., Mertens, F. and Johansson, B. (1997). A breakpoint map of recurrent chromosomal rearrangements in human neoplasia. Nat Genet 15 Spec No: 417-474.
  13. Padilla-Nash, H. M., Heselmeyer-Haddad, K., Wangsa, D., Zhang, H., Ghadimi, B. M., Macville, M., Augustus, M., Schrock, E., Hilgenfeld, E. and Ried, T. (2001). Jumping translocations are common in solid tumor cell lines and result in recurrent fusions of whole chromosome arms. Genes Chromosomes Cancer 30(4): 349-363.
  14. Schueler, M. G., Higgins, A. W., Rudd, M. K., Gustashaw, K. and Willard, H. F. (2001). Genomic and genetic definition of a functional human centromere. Science 294(5540): 109-115.
  15. Yadlapalli, S. and Yamashita, Y. M. (2013). Chromosome-specific nonrandom sister chromatid segregation during stem-cell division. Nature 498(7453): 251-254.

简介

人类着丝粒由重复的α卫星DNA的大串联阵列组成,这些细胞通常是癌症中异常重排的位点(Mitelman等人,1997; Padilla-Nash等人 >,2001)。迄今为止,对人类着丝粒重复序列的注释仍然不完整,极大地妨碍了这些区域对染色体分离至关重要的深入功能研究。为了监测由于重组和诱变事件而在着丝粒(C-SCE)上发生姊妹染色单体交换,我将染色体定位荧光原位杂交(CO-FISH)技术应用于着丝粒( Cen-CO-FISH)在人类细胞中的表达。这种基于杂交的方法包括(1)通过单轮复制掺入核苷酸类似物,(2)新合成的DNA链的酶消化和(3)单链探针的后续杂交,在不存在变性步骤的情况下。所产生的信号允许基于DNA的5'-3'方向性差异地标记每个姊妹染色单体,并评估指示C-SCE的异常染色模式。应用于人类着丝粒的Cen-CO-FISH方法揭示,人类着丝粒确实在循环细胞中发生重组,导致C-SCE,并且在经历衰老的癌细胞系和原代细胞中着丝粒不稳定性增强(Giunta和Funabiki,2017)。在这里,我介绍了人类细胞中Cen-CO-FISH方法的制备,实验程序和数据采集的详细方案。它还包括该技术的概念性概述,以及代表性图像和评分准则的示例。 Cen-CO-FISH是促进着丝粒重复探索的有用工具。

【背景】人类基因组计划于2003年标记为完成,但它遗漏了超过10%的人类重复DNA(de Koning et al。,2011),包括着丝粒。人类着丝粒是一个高度专业化的基因组基因座(Choo,1997),在染色体分离过程中发挥着关键作用,在细胞分裂过程中它作为动粒装配的位点以允许与微管和姊妹染色单体分离相互作用(Cheeseman,2014)。人类着丝粒由称为α-人造卫星的特征性重复DNA序列组成,其线性装配在参考基因组中基本不存在。在这里,我介绍了应用Cen-CO-FISH技术来标记人类着丝粒并监测导致交叉的重组事件。 Bailey等人在20多年前引入了Bailey等人,1996年,CO-FISH方法被广泛应用于检测端粒重复序列的重组,脆性,复制时间,融合和倒位,以及监测有丝分裂分离模式和非随机姐妹染色单体分离(Bailey等人,2010)。这种方法应用于着丝粒,在此称为Cen-CO-FISH方法,已经揭示了着丝粒特异性组蛋白变体CENP-A和CENP-A相关蛋白CENP-C和CENP-T / W的作用是防止着丝粒不稳定性,并且这种功能在癌细胞系和原代细胞中趋于复制衰老,显示更高数量的C-SCE(Giunta和Funabiki,2017)。 Cen-CO-FISH用于评估人类细胞中癌症和细胞衰老过程中的着丝粒不稳定性(Giunta和Funabiki,2017),并且之前已应用Cen-CO-FISH研究重组(Jaco et al。,2008 ; de La Fuente et al。,2015)和小鼠细胞中的姊妹染色单体分离模式(Falconer等人,2010)。该方法的广泛应用潜力来自定量检测α卫星重复,导致C-SCE的着丝粒重组,脆性,复制时间,融合和倒位,以及监测有丝分裂分离模式和非随机姐妹染色单体分离(贝利 ,2010; Yadlapalli和Yamashita,2013)。 Cen-CO-FISH填补了缺失的遗传信息中的空白,这些信息对着丝粒和其他重复区域产生了阴影,为我们基因组这些重要基因座的功能探索带来了新的光芒。

关键字:着丝粒, 荧光原位杂交, CO-FISH, α卫星, 重复DNA, 基因组稳定性, 重组, 姐妹染色单体交换

材料和试剂

  1. 6或10厘米培养皿(Corning,Falcon ,产品目录号:353002或353003)
  2. 15ml Falcon管(Corning,Falcon ,目录号:352097)
  3. 磨砂玻璃(Superfrost Plus; Fisher Scientific,目录号:12-550-15)
  4. 盖玻片(24 x 60毫米)(Fisher Scientific,目录号:12-545-M)
  5. 纸巾
  6. 玻璃巴斯德吸管(Fisher Scientific,目录号:13-678-20A)
  7. 手套和实验室外套
  8. 感兴趣的人类细胞和适当的培养基
    注意:该协议适用于贴壁细胞,可以对非贴壁培养物进行改变。
  9. 5'-溴脱氧尿苷(BrdU)(MP Biomedicals,目录号:100166)
    注意:在双蒸水(1,000x)中制备10mM储备液;等分试样并储存在-20°C。
  10. 5'-溴脱氧胞苷(BrdC)(Sigma-Aldrich,目录号:B5002)
    注意:在双蒸水(1,000x)中制备10mM储备液;等分试样并储存在-20°C。
  11. Colcemid(Roche Diagnostics,目录号:10295892001;已稀释10μg/ ml) - 在4℃储存
  12. 磷酸盐缓冲盐水(PBS)
  13. 胰蛋白酶-EDTA(Thermo Fisher Scientific,Gibco TM,目录号:25300)
  14. 胎牛血清(亚特兰大生物)
  15. 氯化钾(KCl)(Fisher Scientific,目录号:P217-500)
  16. RNase A(Sigma-Aldrich,目录号:R5000)
    注:在10mM Tris-HCl pH7.2中制备50mg / ml储备溶液。分装并储存在-20°C。
  17. Hoechst 33258(Thermo Fisher Scientific,Invitrogen TM,目录号:H3569)
    注意:在双蒸水中制成10μg/ ml溶液,并在4°C远离光线存放。
  18. 核酸外切酶III和缓冲液(Promega,目录号:M1811) - 在-20°C保存
  19. DAPI(Sigma-Aldrich,目录号:D9542)-0.5mg / ml在水中的储液。
    在4°C的黑暗中保存一年
  20. ProLong Gold抗褪色试剂(Thermo Fisher Scientific,Invitrogen TM,目录号:P36934)
  21. 指甲油(莎莉汉森,透明哈德勒)
  22. 甲醇(Fisher Scientific,目录号:A452-4)
  23. 冰醋酸(Fisher Scientific,目录号:A38C-212)
  24. 乙醇100%(Decon,目录号:2716),90%和70%
  25. 阻断剂(Roche Diagnostics,目录号:11096176001)
  26. 马来酸(Sigma-Aldrich,目录号:M0375)
  27. 氯化钠(NaCl)(Merck,目录号:SX0420-5)
  28. 氢氧化钠(NaOH)(Fisher Scientific,目录号:S318-500)
  29. Tris-HCl pH 7.2(Sigma-Aldrich,CAS号:1185-53-1)
  30. 甲酰胺(Fisher Scientific,目录号:BP228;使用去离子进行杂交)
  31. 牛血清白蛋白(BSA)
  32. Tween-20(Hoefer,CAS号:GR128-500)
  33. 柠檬酸钠(Sigma-Aldrich,CAS号:6132-04-3)
  34. 氯化镁(MgCl 2)(Fisher Scientific,目录号:AC41341-5000)
    制造商:Acros Organics,目录号:413410025。
  35. 二硫苏糖醇(DTT)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0861)
  36. 低渗解决方案(请参阅食谱)
  37. 固定剂解决方案(请参阅食谱)
  38. 乙醇稀释(见食谱)
  39. 阻止解决方案(请参阅食谱)
  40. 杂交解决方案(请参阅食谱)
  41. 杂交洗#1(见食谱)
  42. 杂交洗#2(见食谱)
  43. 肽核酸(PNA)探针(来自PNABio的定制探针)(见食谱)
  44. 氯化钠和柠檬酸钠缓冲液(SSC,见食谱)

设备

  1. 移液器(吉尔森)
  2. 离心机(Eppendorf,型号:5810 R)
  3. Coplin Jars(Scienceware,Sigma-Aldrich,目录号:S5641)
  4. 加热块(VWR)
  5. 水浴(Fisher Scientific,型号:Isotemp TM 210)
  6. 具有365-nm紫外线光谱的Stratalinker(Spectralinker XL-1000 1800 UV辐照器)(Spectronics Corporation,型号:XL-1000)
  7. 滑动塑料托盘 - 以适应Stratalinker
  8. 杂交室(详见文本)
  9. 轨道摇床
  10. 成像设备:
    1. DeltaVision图像恢复显微镜系统(Applied Precision / GE Healthcare)
    2. 奥林巴斯IX-70显微镜(奥林巴斯,型号:IX70)
    3. 100x / 1.40 UPLSAPO物镜
    4. CoolSnap QE CCD相机(Photometrics)

软件

  1. SoftWoRx(由Applied Precision出售)
  2. Metamorph 7.8(由Universal Imaging出售)
  3. 棱镜5(由GraphPad出售)

程序

  1. 掺入BrdU:C和中期染色体涂抹制剂
    1. 收获前至少两天将细胞接种在6或10厘米的培养皿中,在收获时约为70%汇合。在一个10厘米的盘子里,种子约有一百万个细胞。
    2. 然后通过与5'-溴脱氧尿苷(BrdU)和5'-溴脱氧胞苷(BrdC)温育标记细胞16-20小时。解冻1,000x BrdU和BrdC的等分试样并混合3:1以获得7.5mM BrdU + 2.5mM BrdC。以1:1,000的稀释度添加到细胞培养基中并孵育细胞过夜。
      注意:根据您的细胞生长较慢/较快以避免双重标记或未完全标记的细胞,调整BrdU:C标记的时间。您的目标是细胞在整个S期中整合BrdU和BrdC,并阻止它们在复制后退出有丝分裂。通常,将HeLa细胞与BrdU:C孵育14-16小时,然后加入秋水仙碱;以验证BrdU:C孵育的时间,可以使细胞与G1 / S上的胸苷同步,通过流式细胞术释放和监测有丝分裂退出。在胸腺嘧啶脱氧核苷释放15小时后退出有丝分裂的细胞应该用BrdU:C处理16-17小时,然后加入秋水仙碱,以确保只有在S期前开始标记的细胞被谷胱甘肽阻滞所捕获。 />
    3. 添加colcemid到最终浓度0.1μg/ ml(1:100稀释10μg/ ml储液)以在收获前累积有丝分裂细胞约2小时。
      注意:避免将细胞留在秋水仙中超过3小时,因为长时间暴露于秋水仙碱会导致染色体形态非常紧凑。
    4. 通过将上清液保存在15ml Falcon管中收获浮动细胞,用PBS洗涤培养皿一次,并将PBS洗涤物收集到相同的管中。胰蛋白酶消化5分钟,用含有胎牛血清的培养基封闭,并将其余的细胞收集在同一管中。
    5. 在175μg×g下沉淀细胞5分钟。去除上清液,并重悬细胞在10毫升预热75毫米氯化钾溶液。轻轻地上下移动以重新悬浮颗粒。
    6. 将细胞在KCl低渗溶液中37°C孵育30分钟(缓慢颠倒管每10分钟以保持细胞悬浮)。在将细胞沉淀之前,加入200μl固定溶液并通过缓慢颠倒管两次混合,有助于中期在离心过程中保持完整,因为低渗处理后细胞非常脆弱。将细胞在175μgxg下沉淀5分钟。
    7. 吸出大部分上清液,留下约200μl的氯化钾通过轻拍管重新悬浮细胞或慢慢用P1000上下吹打。
    8. 通过逐滴加入10ml新鲜制备的冰冷的MeOH-乙酸(3:1)固定剂溶液(在使用前30分钟制备,保持在-20℃),同时在涡旋混合器上连续涡旋该管来固定再悬浮的小球在最低速度设置(#1)。
      注意:重要的是在加入固定剂之前重悬沉淀物,并且一次添加固定剂一滴,同时以至少第一次2ml的低速连续涡旋细胞。其余8毫升可以一次加入。
    9. 将管倒置数次来混合。
    10. 现在细胞可以保持在4°C几个月,远离光线储存。
    11. 一旦准备好放下细胞,将试管在380×g 下旋转5分钟。根据每个试管中有多少个细胞吸出固定剂,留下约0.5-2毫升。
      彻底重新悬浮细胞
    12. 如果您正在使用Superfrost Plus幻灯片,请直接转到步骤A13。如果使用常规载玻片,请在肥皂水中清洗并在使用前用Milli-Q水冲洗3次。将Milli-Q水在冰箱中放置一小时并在使用前保持干燥。
    13. 通过将其放置在置于加热块上的热湿毛巾上,将您即将“降落”的载玻片加湿,直至其蒸发。您也可以通过在呼吸道上“呼吸”来达到此目的。
      注意:创建湿度条件,例如将湿纸巾放在相邻的90°C块上,继续释放蒸气,并且在紧邻滑片落下处加湿空气,或使用设定湿度的混合罩和温度,有助于提高价差的质量。
    14. 使用玻璃巴斯德吸管,上下吸移并吸出细胞悬液。从约20厘米处将几滴细胞悬液倒入载玻片上的不同位置。
      您应该瞄准每次放置滑盖的所有部分。
      关于安全的注意事项:应使用手套和实验服来防止可能的飞溅。整个中期扩散程序可以在实验室化学通风橱中进行,以获得额外的保护。
    15. 滴完后,立即将载玻片放置在42°C的块上,用湿纸巾覆盖块,约2-3分钟,细胞面朝上。
      注意:影响传播的因素有:湿度,温度,细胞滴在载玻片上的距离和干燥时间。
    16. 在室温下远离光线的空气中干燥过夜。您可以将任何储存在新鲜固定剂中的隼管保存在4°C的温度下长达一年。

  2. BrdU:C标记的新合成链的酶消化
    1. 将滴落的载玻片插入Coplin广口瓶中,并在PBS(pH 7.0-7.5)中再水化5分钟(每个罐需要约35ml的溶液)。
    2. 在37℃水浴中用0.5mg / ml RNA酶A(在PBS中,不含DNase)处理载玻片10分钟。
    3. 在室温下轻微搅动下,用1μg/ ml Hoechst 33258(Sigma-Aldrich)稀释至2×SSC 15分钟,染色载玻片。
    4. 将幻灯片放在浅塑料托盘中,并添加足够的2x SSC缓冲液以覆盖幻灯片。在Stratalinker 1800中,在室温下将载玻片暴露于365 nm紫外光下1800秒(30分钟;相当于5,400 J / m 2 2)。
    5. 将BrdU:C标记的UV切口DNA链与80μl10U /μl核酸外切酶III(Promega)一起加入制造商提供的缓冲液中,稀释至1x(50mM Tris-HCl,5mM MgCl 2, ,5mM DTT,pH 8.0)。将80μl溶液加到24×60盖玻片上,该盖玻片将与载玻片上的细胞的整个表面接触。让它粘附在载玻片上,然后将载玻片的一面朝上放置10分钟,远离光线。避免气泡。
    6. 去除盖玻片并用新鲜溶液重复核酸外切酶III消化步骤10分钟。
    7. 在PBS中清洗载玻片5分钟,并依次在70%,90%,100%乙醇系列中各自脱水2分钟。
    8. 空气干燥的幻灯片和在室温下在黑暗中(可以放置过夜)。

  3. 荧光原位杂交
    1. 将80μl杂交混合物放到盖玻片上,并用载玻片取出盖玻片,然后将载玻片放入杂交室(参见图1G-1I关于如何制备杂交室的说明以及这些步骤的视觉说明)。避免气泡。


      图1.在协议不同阶段的Cen-CO-FISH程序的代表性图像。 :一种。收获细胞,低渗肿胀并固定过夜。样品可以在此阶段储存在4°C。然后将细胞滴在载玻片上以使中期分裂。在进入步骤B之前,可以在室温(RT)下在黑暗中储存载玻片。B.将载玻片在Coplin广口瓶中再次水合PBS 5分钟。 C.去除PBS并将RNA酶A溶液加入罐中,在37℃水浴中孵育10分钟。去除RNase溶液并在室温下用2x SSC与Hoechst孵育15分钟。 D.将幻灯片放入塑料试片并用足够的2x SSC覆盖。 E.将托盘放入带有365纳米紫外线灯的Stratalinker烤箱中并暴露30分钟(1,800秒)。 F.为每张载玻片准备80μl的核酸外切酶III溶液,并将溶液加入24×60盖玻片中,使其与载玻片的整个表面接触。 G.紧接紫外线照射后,如图所示用核酸外切酶III溶液提取盖玻片。翻转载玻片,调整盖玻片以确保它是中心的,液体分布良好并且没有气泡。室温孵育细胞10分钟,然后再次重复步骤F-G进行额外的核酸外切酶温育。用PBS洗涤并在乙醇系列中脱水。在室温下在黑暗中存放载玻片(过夜)。 H.将其中一种探针(1:1,000-1:5,000)置于杂交溶液中,在60°C下加热10分钟,然后将80μl加入到盖玻片上。按照所示的步骤F-G继续。 I.将载玻片放入杂交室并在室温孵育2小时。做一个房间,拿一个盒子,用纸巾填满,然后湿润,直到所有的毛巾潮湿。不要将幻灯片直接放在湿纸上面。使用移液器或其他形式的支撑物将盖玻片抬到湿纸上。将幻灯片的单元面朝上放置并远离光线将其关闭。 2小时后打开箱子,你会看到盖子上有少量凝结物。用杂交缓冲液#1洗涤并用第二个反向补体探针重复步骤H(可以颠倒正向和反向补体探针之间的杂交顺序,它应该产生相同的结果)。在杂交缓冲液#1和#2中洗涤,包括DAPI步骤,脱水并风干至少1小时。在成像之前安装并密封。

    2. 杂交室在黑暗中以1:1,000稀释度与正向PNA探针杂交(在使用前在60℃加热10分钟),然后在杂交室中避光2小时。
    3. 冲洗1号洗涤30秒。
      保持幻灯片垂直排水10秒。
    4. 与反向互补PNA探针以1:1,000稀释度(在使用前在60℃加热10分钟)在黑暗中杂交2小时。
      注意:颠倒探针的顺序不会影响信号的杂交或质量。

    5. 在定轨摇床上用Wash#1洗板1次15分钟2次
    6. 在#2号洗涤液中洗涤5分钟3次。第二次洗涤后,从0.5 mg / ml储液中加入1:500 DAPI。

    7. 在70%,90%,100%乙醇系列中脱水3分钟
    8. 空气干燥幻灯片约1小时。
    9. 使用Prolong Gold嵌入介质(避免气泡)装载幻灯片并使用指甲油密封。幻灯片已准备好进行成像。
    10. 幻灯片可在4°C或-20°C下保存1周,以保存更长时间。 

数据分析

图像采集和处理

  1. 使用安装在Olympus IX-70显微镜上的DeltaVision图像恢复显微镜系统(Applied Precision / GE Healthcare)对细胞进行成像,并装配100x / 1.40 UPLSAPO物镜和CoolSnap QE CCD相机(Photometrics)。
  2. 对每个实验的15个或更多中期扩展进行成像以产生统计学显着和代表性的结果。对细胞进行成像时,应注意选择染色体分离良好的最佳涂片(图2A),在核型稳定的二倍体细胞的情况下应选择约46条染色体。 DAPI的典型曝光时间为0.3秒,因为TRITC通道(红色探针)为0.1秒,而FITC(绿色探针)为0.5秒。
  3. 该图像是作为包含约15-30个0.2μm部分的z-叠层获取的。采用SoftWoRx(Applied Precision)对获取的图像进行解卷,并将其导出至Metamorph(Universal Imaging),以进行最大投影分析,如图2A和2B中的示例所示。

代表性图片和评分

  1. 按照概述的Cen-CO-FISH方案,如图2A所示,用特定的着丝粒标记物获得高质量的中期扩散。标记为红色的正向探针和标记为绿色的反向链探针不应重叠,而是彼此相邻,标记每个染色体的一个姐妹染色单体(图2B)。通过计算每个中期扩散中指示C-SCE的异常Cen-CO-FISH模式的数量进行量化(图2A,彩色框)。显示了正常(顶部面板,蓝绿色盒子;图2B)和指示C-SCE的异常Cen-CO-FISH模式的例子(底部面板,红色框;图2B)。在高染色体不稳定性(CIN)系或一些癌细胞系中,由着丝粒区外发生的融合事件可以发现少量异常模式,如通过分离两对着丝粒信号的DAPI的存在所指示的,以及应该从C-SCE的量化中排除(灰色框;图2C)。如先前所示(Giunta和Funabiki,2017),C-SCE的数量可表示为异常着丝点与每个中期相关的标记着丝粒对总数的比例,并使用Prism绘制在散点图中。


    图2.使用Cen-CO-FISH方法检测人类着丝粒处的重组事件。 :一种。使用针对CENP-B盒序列产生的正向(红色)和反向(绿色)探针,在存在于A中显示的中期扩散的所有染色体中的Cen-CO-FISH对HeLa细胞进行染色。单个通道允许目测识别异常Cen-CO-FISH染色模式,通常作为红色通道的“双点”和绿色通道的相同,如白色箭头所示。还显示了与DAPI合并的彩色组合图像。图2B中的框放大了。比例尺= 5微米。 B.来自图2A的左侧面板的放大的盒子,左侧面板显示用于正常Cen-CO-FISH染色模式(以深青色标记)的代表性图像,以及下面三个面板显示指示C-SCE的异常模式(标记为红色)。比例尺= 1μm。 C.由着丝粒序列外重组产生的异常染色模式(以灰色标记)的实例,因此排除了对着丝粒重组和C-SCE的定量。比例尺= 1μm。

  2. Cen-CO-FISH是一种可靠,可重复和灵敏的标记技术,用于可视化重复的基因组区域。该技术依赖于两个关键步骤:位点的重复性以及将BrdU和BrdC并入新合成的链中。用于Cen-CO-FISH杂交的探针是荧光标记的PNA 17或18-mer。设计针对重复序列的探针可以扩增荧光强度。 Centromere alpha卫星重复序列从0.5-5 Mb到每个人类染色体共同的序列(Schueler等人,2001; Fukagawa,2004),产生一个可靠的Cen-CO-FISH信号用于可视化,获取使用传统的显微镜和功能分析。该技术依赖于新合成的链的标记,随后将被酶促消化,这表明该方法仅可用于循环细胞。然而,这也表示这种方法的优点,其中所产生的模式指示在单个先前细胞周期期间发生的重组事件。因此,Cen-CO-FISH提供了这些黑暗基因组区域的鸟瞰图,同时给出了特定的时间分辨率和序列方向信息来检查着丝粒重复的行为。先前的研究报道了应用Cen-CO-FISH技术来监测小鼠细胞中的着丝粒重组(Bailey等人,1996; Jaco等人,2008; Falconer等人,2010)表明该技术可以成功地应用于来自不同物种的组织培养细胞,使用专门设计的探针。

食谱

  1. 低渗解决方案
    75毫摩尔KCl

    每次使用前在37°C水浴中预热所需量
  2. 固定解决方案
    3份甲醇
    1份冰醋酸

    每次都必须新鲜 使用冰冷的
  3. 乙醇稀释

    在双蒸水中70%和90%
  4. 阻止解决方案

    将封闭剂(罗氏)溶解在马来酸缓冲液中制成10%的储备液 100毫摩尔马来酸
    150 mM NaCl
    用NaOH将pH调节至7.5,并保存在4°C
    每次使用前都要剧烈摇动
  5. 杂交解决方案
    10 mM Tris-HCl pH 7.2
    70%甲酰胺
    0.5%封闭剂(来自10%库存)
  6. 杂交洗#1
    10 mM Tris-HCl pH 7.2
    70%甲酰胺(Sigma-Aldrich)
    0.1%BSA(在加入甲酰胺前溶于双蒸水中)
  7. 杂交洗#2
    0.1M Tris-HCl pH 7.2
    0.15M NaCl
    0.1%Tween-20
    对于DAPI清洗,使用0.5 mg / ml的原液添加1:500 DAPI。
  8. 肽核酸(PNA)探针(来自PNABio的定制探针)

    对alpha卫星设置#1 F3003 CENT-Cy3(Cy3-OO-AAACTAGACAGAAGCATT)
    反向互补CENT-RC-488(Alexa488-O-AATGCTTCTGTCTAGTTT)
    在CENP-B框中设置#2:
    F3002 CENP-B Cy3(ATTCGTTGGAAACGGGA)
    反向补充CENP-B-RC-488(TCCCGTTTCCAACGAAT)
    注意:
    1. 基于制造商的说明将冻干的探针在双蒸水中稀释至50μM。分装并储存在-80°C以避免多次冻融。
    2. 为了杂交,解冻并旋转探针,并以1:1,000-1:5,000的稀释度稀释到杂交溶液中(根据获得的信号进行调整)。使用前在60°C加热10分钟。
  9. 氯化钠和柠檬酸钠缓冲液(SSC; 20x)
    NaCl(3M),柠檬酸钠(300mM)溶解在蒸馏水中
    调整pH值到7.0并且高压灭菌器
    工作稀释是2次

致谢

我要感谢洛克菲勒大学生物影像资源中心(BIRC)的H. Funabiki,F. Lottersberger,N. Bosco和T. DeLange,以及K. Thomas,A. North和T. Tong。我还要感谢Seneca Jason和A. Field在整个工作中的支持。这项工作得到了美国 - 意大利癌症基金会的奖学金和洛克菲勒科学奖学金的资助。该协议是根据Giunta和Funabiki(2017)和Celli等人发表的程序改编的。 (2006)。
竞争利益声明:作者声明不存在利益冲突。

参考

  1. Bailey,S.M.,Goodwin,E.H。,Meyne,J.and Cornforth,M.N。(1996)。 CO-FISH揭示与等染色体形成相关的倒位。 诱变 11(2):139-144。
  2. Bailey,S.M.,Williams,E.S。,Cornforth,M.N。和Goodwin,E.H。(2010)。 染色体定位荧光原位杂交或链特异性FISH 方法Mol Biol 659:173-183。
  3. Celli,G.B.,Denchi,E.L。和de Lange,T。(2006)。 Ku70刺激功能异常的端粒融合,但保护染色体末端免受同源重组。 Nat Cell Biol 8(8):885-890。
  4. Cheeseman,I. M.(2014)。 动粒。 冷泉Harb Perspect Biol 6(7):a015826。
  5. Choo,K. H. A.(1997)。着丝粒。 牛津大学出版社,美国。
  6. de Koning,A.P.,Gu,W.,Castoe,T.A.,Batzer,M.A。和Pollock,D.D。(2011)。 重复元素可能占人类基因组的三分之二以上。
  7. de La Fuente,R.,Baumann,C。和Viveiros,M.M。(2015)。 ATRX导致小鼠胚胎母体基因组中主要卫星转录物的表观遗传学不对称和沉默。 开发 142(10):1806-1817。
  8. Falconer,E.,Chavez,E.A.,Henderson,A.,Poon,S.S.,McKinney,S.,Brown,L.,Huntsman,D.G。和Lansdorp,P.M。(2010)。 DNA模板链序列鉴定姊妹染色单体。 Nature 463(7277):93-97。
  9. Fukagawa,T。(2004)。 脊椎动物细胞中的着丝粒DNA,蛋白质和动粒组装。 Chromosome Res 12(6):557-567。
  10. Giunta,S.和Funabiki,H。(2017)。 人类着丝粒DNA重复序列的完整性受CENP-A,CENP-C和CENP- T. 美国国家科学院院刊114(8):1928-1933。
  11. Jaco,I.,Canela,A.,Vera,E。和Blasco,M.A。(2008)。 哺乳动物细胞中的着丝粒有丝分裂重组。
  12. Mitelman,F.,Mertens,F.和Johansson,B。(1997)。 人类瘤形成中复发性染色体重排的断点图。 Nat Genet 15规格编号:417-474。
  13. 帕迪拉 - 纳什,HM,Heselmeyer-Haddad,K.,Wangsa,D.,Zhang,H.,Ghadimi,BM,Macville,M.,Augustus,M.,Schrock,E.,Hilgenfeld,E。和Ried,T (2001)。 跳转易位在实体瘤细胞系中很常见,并导致整个染色体臂复发性融合。 基因染色体癌症 30(4):349-363。
  14. Schueler,M.G.,Higgins,A.W.,Rudd,M.K。,Gustashaw,K.and Willard,H.F。(2001)。 功能性人类着丝粒的基因组和基因定义。 Science 294(5540):109-115。
  15. Yadlapalli,S.和Yamashita,Y.M。(2013)。 干细胞分裂过程中染色体特异性非随机姐妹染色单体分离。 Nature 498(7453):251-254。
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引用:Giunta, S. (2018). Centromere Chromosome Orientation Fluorescent in situ Hybridization (Cen-CO-FISH) Detects Sister Chromatid Exchange at the Centromere in Human Cells. Bio-protocol 8(7): e2792. DOI: 10.21769/BioProtoc.2792.
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