To develop these protocols, human oocytes and blatomeres are obtained after informed consent from women participating in in vitro fertilization (IVF) programs. The oocytes used are those that fail to fertilize and to cleave after in vitro insemination. Embryos used are donated embryo that have abnormal development and are not involved in IVF procedure. Single blastomeres are obtained from biopsy or by disaggregation of eight-cell-stage human embryo. Isolated oocytes and blastomeres are kept in drops of medium covered with silicone oil, in culture Petri dishes stored into an incubator at 37°C, 5% CO2, and 95% humidity.
1. Using a micropipet, transfer individual oocyte from the culture medium droplet to hypotonic solution, under the control of a dissecting microscope (see Note 1).
2. The cell is exposed to hypotonic solution for approx 3 to 5 min under the control of the dissecting microscope. Check cell to make sure it has swollen 1.5 to 2 times its original size.
3. Carefully transfer the cell onto a microscopic slide in a 1- to 2-|L drop of hypotonic solution.
4. Using an automatic pipettor set at 17 |L, drop one drop of fresh fixative directly on citrate drop from a height of approx 2 cm (see Note 2).
5. As the drop starts to spread out on the slide, the cell will become quite visible under the fluorescent light, standing out in relief. A very gentle moist breath can be used to stop cell from rolling around the slide. This will add moisture to the slide and stabilize the cell. Use then a diamond marker to circle the cell on the underside of the slide.
6. Immediately add a second drop of fixative above the area where the cell was circled. Do not aJJow the slide to dry. The drop will spread out along the slide toward the edges and begin beading (see Note 3).
7. Add a third drop of fixative onto the cell. Do not allow the slide to dry.
8. When rainbow effect appears, add a fourth drop of fixative.
9. At this point, when the drop beading stops and the rainbow effect appears, use a long, gentle dry breath to dry the last fixative from the slide.
10. Using phase contrast microscope, check within the circle to ensure that nucleus or chromosomes are present.
11. Dehydrate the slide in an ethanol series (70%, 90%, 100%), 2 min each step, and air-dry.
12. Store the slide at room temperature in a hermetic box in order to avoid dust deposits.
13. Before the PRINS reaction, denature chromosomal DNA by immersing the slide in 70% formamide, 2X SSC, pH 7.0, at 73°C for 3 min.
14. Pass the slide through an ice-cold ethanol series (70%, 90%, 100%), 2 min each step, and air-dry.
3.1.2. Human Blastomere Preparation
It is possible to fix isolated blastomeres by using the above described fixation technique.
(Subheading 3.1.1., steps 1 to 12). As an alternative, the following method can also be used (see Note 4):
1. Prepare fresh spreading solution by mixing 1 mL of 1% Tween-20 solution, 0.1 mL of 1 NHCl, and 8.9 mL of distilled water
2. Make a circle on the underside of a poly-L-lysine-coated slide using a diamond marker. Put a small drop of hypotonic solution on the slide in the circle and a small drop of 1X PBS in one corner of the slide.
3. Using a micropipet, transfer the isolated blastomere from the culture medium drop to the drop of PBS on the slide, under the control of a dissecting microscope.
4. Using a micropipet, transfer the cell into the drop of hypotonic solution within the circle, ensuring minimum transfer of PBS.
5. Before complete drying of the drop (approx 1-2 min), add continuously spreading solution to the drop until the cell starts to lyse (see Note 5). With constant observation of the cell, keep replacing the spreading solution until the lysis of the cell membrane. The spreading solution must not be allowed to dry out before the complete cell lysis.
7. When the cell is lysed and the cytoplasm washed away from the nucleus, leave the slide to air-dry and incubate in 1X PBS for 5 min.
8. Dehydrate the slide in an ethanol series (70%, 90%, 100%), 2 min each step, and air-dry.
9. At this stage, the slide can be stored for up to 1 wk at room temperature in a hermetic box (to avoid dust deposits).
10. Before the PRINS reaction, prepare a 0.01 NHCl solution in a Coplin jar and heat to 37°C in water bath. Add a 0.5-mL aliquot of 10 mg/mM pepsin to HCl and mix well.
11. Place the slide in this 1% pepsine solution and incubated the slide at 37°C for 3 to 10 min, depending on the amount of remaining cytoplasm (see Note 6).
12. Rinse briefly the slide on IX PBS.
13. Wash the slide twice 1 min in distilled water.
14. Pass the slide through an ethanol series (70%, 90%, 100%), 2 min each step, and air-dry.
15. Denature chromosomal DNA by immersing the slide in 70% formamide; 2X SSC, pH 7.0; at 73°C for 3 min.
16. Pass the slide through an ice-cold ethanol series (70%, 90%, 100%), 2 min each step, and air-dry.
3.2. Multicolor PRINS (on Oocyte or Blastomere Preparations)
In the three-color PRINS procedure, three sequential PRINS reactions are performed, each labeling one specific chromosome. The following labeling order is used (see Note 7): (1) FITC for the first targeted chromosome, 92) TRITC for the second targeted chromosome, and (3) FITC for the third targeted chromosome. In the four-color procedure, this labeling order is supplemented with a fourth PRINS reaction using Cascade Blue (see Fig. 1, Chapter 6).
1. Prepare a reaction mixture for the first PRINS reaction, in a final volume of 50 pL containing: 0.2 mM dATP, dCTP, and dGTP, 0.02 mM dTTP, 0.02 mM FITC-12-dUTP, 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 0.01% BSA, 200 pmol of oligonucleotide primer, and 2.5 U Taq DNA polymerase. In practice, mix in a sterile microcentrifuge tube: 1 pL of each 1:10 diluted dATP, dCTP, and dGTP, 1 pL of the 1:100 diluted dTTP, 1 pL of FITC-12-dUTP, 1 pL of BSA, 5 pL of 10X Taq buffer, 0.5 pL of the Taq DNA polymerase, 4 pL of the specific primer (for instance, the primer specific for chromosome 1 according to the procedure illustrated in Fig. 1, Chapter 6), and distilled water to 50 pL.
2. Place the reaction mixture under a 22 x 32 cover slip on the denatured slide, and transfer to the heating block of the thermal cycler.
3. Set up the PRINS program and start the reaction. The program consists of a unique 5- to 10-min step at the specific annealing temperature of the primer involved for both in situ annealing and elongation.
4. While this first reaction is running, prepare the reaction mixture for the second PRINS reaction as described previously but instead incorporate the specific primer for the second targeted chromosome (for instance, chromosome 9 according to Fig. 1, Chapter 6), and TRITC-6-dUTP.
5. On completion of the program, carefully remove the cover slip from the slide.
6. Wash the slide twice for 2 min at room temperature in 1X PBS.
7. After draining the excess 1X PBS off the slide, and before the slide is completely dry, put the second PRINS reaction mixture on the slide, and cover with a 22 x 32 cover slip.
8. Place the side again on the plate of the thermal cycler.
9. Set up the program for the second PRINS reaction: 5 min at the annealing temperature, specific to the second primer used.
10. Start the program.
No additional denaturation is required after the first PRINS reaction because DNA remains denatured through the PRINS incubations.
11. While this second reaction is running, prepare the reaction mixture for the third PRINS reaction, incorporating the specific primer for the third targeted chromosome (for instance chromosome 16 according to Fig. 1, Chapter 6) and FITC-12-dUTP.
12. At the end of the second reaction, remove the cover slip from the slide and repeat the washing steps 6 and 7.
13. Before the slide is completely dry, put the third PRINS reaction mixture on the slide, and cover with a 22 x 32 cover slip.
14. Place the slide on the thermal cycler.
15. Set up the program for the third PRINS reaction: 5 min at the annealing temperature, specific to the third primer used.
17. At the end of this third reaction, the slide is transferred to 4X SSC, 0.05 Tween-20 for two washes (3 min each) at room temperature, with gentle agitation.
In the four-color procedure, the third reaction is followed by a fourth reaction with the primer specific for the fourth targeted chromosome (for instance chromosome 18 as indicated in Fig. 1, Chapter 6). No additional denaturation is needed.
1. Prepare the reaction mixture for the fourth PRINS reaction, incorporating the specific primer for the fourth targeted chromosome and Cascade Blue-7-dUTP.
2. At the end of the reaction, remove the cover slip from the slide.
3. Transfer the slide in 4X SSC, 0.05 Tween-20 for two washes (3 min each) at room temperature, with gentle agitation.
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