Measurement of Telomere Length Using QFISH

Conducting FISH using a telomere DNA probe (CCCTAA)3 localizes telomeric DNA to bovine metaphase chromosomes (55). The use of Q-FISH, alongside standard curves of fluorescent intensity of telomeric DNA of known length, can determine telomere lengths of individual chromosomes from metaphase nuclei (60). To measure the average length of telomere repeats in individual cells a flow cytometry method using fluorescence in situ hybridization (flow FISH) has also been developed (61).

The underlying principle for the FISH methods of telomere length determination is the quantification of a fluorescent signal obtained from a telomere-specific probe labeled with a flourochrome. To ensure specificity of binding to the telomeric repeat (TTAGGG)n region of the chromosome an 18-bp probe consisting of (AATCCC)3 is used. To increase hybridization efficiency and specificity, the probes for Q-FISH are constructed using a synthetic DNA/RNA analog, PNA. In PNA probes, the sugar phosphate backbone characteristic of nucleic acid probes is replaced by a neutral peptide/polyamine backbone that keeps an even distance between the bases and makes the probe highly resistant to degradation by DNases, RNases, proteinases, and peptidases. PNA probes labeled with FITC or Cy3 for telomere detection can be obtained from various commercial sources (e.g., PE Biosystems, Framingham MA), or telomere PNA FISH kits are also available (e.g., DakoCytomation).

The use of Q-FISH to study telomeres allows the examination of individual chromosomes in single cells (see Fig. 4). Although labor-intensive, it permits the detection of chromosome and/or cell-specific variation. This technique is of particular use when applied to small samples, such as pre-placental stage embryos, which yield too little DNA for telomere length determination by southern blot analysis (TRF method). This protocol comprises three essential steps: chromosome preparation, in situ hybridization, and densitometry.

3.4.1. Chromosome Preparations

3.4.1.1. Air-Dried Chromosome Spreads From Lymphocytes and Solid Tissues

1. Establish lymphocyte or whole blood culture according to standard protocols (see Note 9).

2. 1 to 2 h before terminating the culture, introduce a mitotic inhibitor (e.g., colcemide at a final concentration of 0.05 |j.g/mL).

3. Transfer cultures to centrifuge tubes and centrifuge at 100g for 2 to 5 min to concentrate the cells.

4. Remove all but 1 mL of medium and resuspend cells in 10 to 15 mL of 37°C hypotonic (0.075 M) KCl. Let stand for 10 to 15 min. Note that the volume of KCl and the duration should be optimized for each cell type and species.

5. Centrifuge tubes at 100g for 10 min or until a pellet of cells appears on the bottom.

6. Remove all but 0.5 mL of KCl and resuspend the cell pellet in freshly prepared methanol/acetic acid fixative (3/1 v/v) and let stand for 30 min.

7. Centrifuge at 100g for 10 min, discard fixative and replace with fresh fixative. Cell suspensions can be stored overnight at 4°C or for long periods at -20°C.

8. The day before hybridization, centrifuge the cells to form a pellet, remove the old fixative and resuspend the cells in a small volume of freshly prepared fixative (0.5-2 mL).

9. Drop cell suspension on precleaned slides to allow metaphase chromosomes to spread. Air dry the slides overnight. Note the volume of fixative in step 8 should be adjusted to the size of the pellet to ensure those chromosomes are well spread out with few overlapping chromatids.

3.4.1.2. Chromosome Spreads for Tissue Cultures

1. Establish and maintain tissue cultures according to protocol of choice.

2. When cells are in rapid growth phase introduce a mitotic inhibitor (e.g., colcemid at a final concentration of 0.05 |g/mL) to the culture vessels.

3. Continue incubation for 1 to 4 h.

4. Detach dividing cells by gentle trypsinization and agitation.

5. Centrifuge and proceed as for lymphocytes above beginning at step 3.

3.4.1.3. Air-Dried Chromosome Spreads From Zona Pellucida

Enclosed Oocytes and Embryos

1. Introduce embryos to equilibrated prewarmed culture medium containing a microtubule inhibitor such as colcemid at a final concentration of 0.05 |g/mL medium (see Note 10).

2. Incubate at 39°C in 5% CO2 for up to 5 h (minimum of 30 min).

3. Transfer the embryo into a hypotonic solution (0.88% sodium citrate) and keep at room temperature for 3 to 5 min (requires optimization).

4. Place the embryo with a small drop of hypotonic solution onto a pre-cleaned slide. The hypotonic solution should not be allowed to evaporate as this will dry the embryo or (oocyte) and prevent spreading of the chromosomes.

5. Drop one to two drops (~50 |L) of freshly-prepared fixative onto the embryo (1/ 1 methanol/acetic acid for bovine embryos and 3/1 for mouse embryos)

6. Mark a circle on the back of the slide to indicate the location of the embryo.

7. Air-dry by gently blowing on the slide.

8. Place the slide into fresh fixative (3/1 methanol/acetic acid) for 30 min (if bovine embryos).

9. Remove the slide from the fixative and air-dry at room temperature in a fume hood or well-ventilated area.

3.4.2. In Situ Hybridization and Image Analysis

1. Prepare the slides the day before FISH and let them dry overnight at room temperature.

2. Rehydrate the cells on the slides in 1X PBS (Ca2+ and Mg2+ free, pH 7.0-7.5,) for 15 min.

3. Fix in 4% formaldehyde in 1X PBS for 2 min.

5. Treat with 1 mg/mL pepsin at 37°C for 10 min (note: pepsin should be prepared freshly in acidified water pH 2.0).

6. Wash twice in 1X PBS for 2 min.

7. Fix in 4% formaldehyde in 1X PBS for 2 min.

8. Wash three times for 5 min in 1X PBS.

9. Serially dehydrate the cells in ethanol: 5 min 70%, 5 min 90%, 5 min 100%.

10. Air dry the slides.

11. Prepare the hybridization mixture:

Stock

Final concentration

For 250 mL

Formamide (ultrapure, pH 7.0-5.0)

70%

175 |L

Blocking protein in water 2.5%

0.25%

25 | L

0.2 M Tris

10 mM

2.5 | L

PNA Telomere—FITC/Cy3 probe

0.5 |g/mL

2.5 |L (100X stock)

MgCl2 buffer, pH 7.4

5%

21.4 |L

dd water

till 250 | L

12. Pipet two drops of 10 ||L of hybridization mix onto a cover slip (24 x 55 mm), cover carefully with the slide (upside down), avoiding air bubbles, and turn the slide over.

13. Denature in a preheated oven (80°C) for 3 min

14. Hybridize in a humidified chamber at room temperature for 2 h.

15. Remove the coverslip carefully in wash solution I (70% formamide, 10 mM Tris, 0.1% bovine serum albumin, pH 7.0-7.5).

16. Wash the slides twice for 15 min in wash solution I.

17. Wash three times for 5 min in wash solution II (0.1 M Tris, 0.15 M NaCl, 0.08% Tween-20, pH 7.0-7.5).

18. Serially dehydrate the cells in ethanol: 5 min 70%, 5 min 90%, and 5 min 100%.

19. Air dry the slides.

20. Place 20-|L drops of Vectashield containing 200 ng DAPI on the coverslip.

21. Keep the slide in a light-protected storage box at 4°C.

22. View under an epifluorescent microscope at a wavelength of 405 and 546 nm, which are used to excite the DAPI (chromosomes) and Cy3 probes (telomeres) respectively, and capture images separately with a CCD monochromatic camera.

23. Digitally captured images of telomeres are then analyzed to estimate telomere length by a variety of image analysis programs (e.g., TFL-TELO [60]; Optimas image analysis software (Media Cybernetics, Silver Spring, MD; [62]), that are designed to compare selected pixel densities identified (see Fig. 4 and Note 11).

Fig. 4. Telomere length measurements using Q-FISH of ovine metaphase chromosomes. (A) Punctate fluorescent signals localized to the end regions of both the long and short arms of both chromatids of each ovine chromosome. (B) Enlarged section of (A) showing a typical fluorescence image of the telomeres and representative segmentation boundaries (C) that are generated for telomere fluorescent intensity quantification. For more information of telomere length measurement using digital fluorescence microscopy, see ref. 60.

Fig. 4. Telomere length measurements using Q-FISH of ovine metaphase chromosomes. (A) Punctate fluorescent signals localized to the end regions of both the long and short arms of both chromatids of each ovine chromosome. (B) Enlarged section of (A) showing a typical fluorescence image of the telomeres and representative segmentation boundaries (C) that are generated for telomere fluorescent intensity quantification. For more information of telomere length measurement using digital fluorescence microscopy, see ref. 60.

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