Introduction

Among all the adaptations of molecular cytogenetic procedures, the in situ chromosomal analysis of isolated human oocytes and blastomeres constitutes an important challenge because schromosomal abnormalities are known to be the major reason for before and after implantation human embryo wastage, with a significant prevalence of female meiotic segregation errors. To date, fluorescence in situ hybridization and primed in situ labeling (PRINS) have been successfully adapted on isolated human gametes (1-3). Because of its relative simplicity and the commercial availability of numerous DNA probes, fluorescence in situ hybridization has become the standard technique for in situ chromosomal investigations. However, the PRINS reaction offers a fast alternative approach based on the use of short, unlabeled, and chromosome-specific primers (4). Primers are annealed in situ to complementary DNA targets on denaturated cell preparations, and then act as primers for chain elongation catalyzed by a Taq DNA polymerase in the presence of free nucleotides. The visualization of generated fragments results from the in situ incorporation of one labeled nucleotide.

From: Methods in Molecular Biology, vol. 334: PRINS and In Situ PCR Protocols, Second Ed. Edited by: F. Pellestor © Humana Press Inc., Totowa, NJ

In the conventional PRINS procedure, the identification of several chromosomes is performed by in situ sequential labeling of each targeted chromosome, using chromosome-specific primers and different reporter molecules or fluorochromes. Between each labeling reaction, a intermediate reaction based on the use of ddNTP is performed to block the free 3'-ends of the generated elongation fragments, and then to prevent the mixing of the labeling (5). This intermediate reaction significantly extends the total duration of the multi-PRINS procedure and may lead to a prejudicial decrease in the intensity of the labeling of the first targeted chromosomes.

Recently, a new multicolor PRINS method has been introduced (6) that eliminates the blocking reaction. This original procedure, based on the in situ mixing of two fluorochromes for generating the distinct labeling of three chromosomes, greatly simplifies the multicolor PRINS protocol and increases the homogeneity of the chromosome labeling. We have adapted and tested this new PRINS protocol on isolated human oocytes and blastomeres.

2. Materials

2.1. Human Oocyte and Blastomere Preparation

1. Phosphate-buffered saline (PBS; Gibco BRL, Eragny, France).

4. Glacial acetic acid (Prolabo).

5. Desionized formamide, stored at 4°C (Intergen Company, New York, NY).

6. 20X standard saline citrate (SSC): 3 MNaCl, 0.3 Mtrisodium citrate, pH 7.5 (can be stored for several months at room temperature).

7. Bovine serum albumin (BSA; Sigma, St. Louis, MO).

8. Distilled water.

9. 1 N HCl solution: dilute 1 mL of pure HCl (Prolabo) with 11 mL of distilled water. Store at 4°C.

10. Hypotonic solution: 1% Na citrate solution (Prolabo) in 0.2 mg/mL BSA.

11. Tween-20 (Roche Diagnostics).

12. Spreading solution: 0.01 NHCl, 0.1% Tween-20. Mix 1 mL of 1% Tween-20 with 0.1 mL of 1 N HCl solution and 8.9 mL of distilled water. Make spreading solution fresh.

13. Pepsine (Sigma): prepare a 10 mg/mL pepsine solution. Dilute 100 mg of pepsine in 10 mL of distilled water. Store frozen in 0.5-mL aliquots. Do not refreeze and only store for 1 to 2 mo.

15. Twin-frost glass microscope slides (CML, Nemours, France). The slides must be cleaned by soaking in absolute ethanol to which concentrated HCL have been added at the rate of 1 mL/100 mL. The slides are removed from the acid/alcohol and polished with clean piece of muslin just before dropping the sperm suspension.

16. Poly-L-lysine-coated slides: dilute 1:10 poly-L-lysine in water, pour into a Coplin jar, and incubate glass microscope slides for 5 min. Leave slides to dry at room temperature overnight and store in fridge.

18. 25- to 50-pL micropipets (Drummond, Broomall, PA) attached to rubber tubing and mouthpiece.

19. 10- to 20-pL automatic pipetor.

20. Diamond marker.

22. Phase contrast microscope with x10, x40, x60 magnifications (Leica France, Rueil-Malmaison, France).

23. Dissecting microscope equipped with a zoom system (Leica).

2.2. Multicolor PRINS

1. dATP: 100 mM solution (Roche Diagnostics, Meylan, France) diluted 1:10 with sterile distilled H2O.

2. dCTP: 100 mM solution (Roche Diagnostics) diluted 1:10 with sterile distilled H2O.

3. dGTP: 100 mM solution (Roche Diagnostics) diluted 1:10 with sterile distilled H2O.

4. dTTP: 100 mM solution (Roche Diagnostics) diluted 1:100 with sterile distilled H2O.

5. Fluorescein isothiocyanate 2'-deoxyuridine 5'-triphosphate (FITC-12-dUTP) 1 mM (Roche Diagnostics).

6. Tetramethylrhodamine isothiocyanate (TRITC-6-dUTP) 1 mM(Roche Diagnostics).

7. Cascade Blue-7-dUTP 1 mM (Molecular Probes, Leiden, The Netherlands).

10. Taq DNA polymerase (Roche Diagnostics) or Ampli Taq (Perkin Elmer, Foster City, CA).

11. 10X Taq buffer: 500 mMKCl, 100 mMTris-HCl, pH 8.3, 15 mMMgCl2.

12. Oligonucleotide primers at 50 pmol/pL (see Table 1, Chapter 6).

13. Sterile distilled water.

14. Tween-20 (Roche Diagnostics).

15. Washing buffer (diluted from 20X SSC): 4X SSC, 0.05% Tween-20.

16. 1.5-mL Sterile microcentrifuge tubes (Eppendorf AG, Hamburg, Germany).

19. Programmable thermal cycler equipped with a flat pate block (Hybaid Ltd., Teddington, UK).

2.3. Detection and Microscopy

2. Propidium iodide (Sigma).

3. Antifade solution Vectashield (Vector Labs, Burlingame, CA).

5. Rubber cement (Artos, Strasbourg, France).

6. Epifluorescence Microscope Leica DMRB (Leica France) equipped with x40 and x100 Plan FluoTar objectives, and with a DAPI single band-pass filter (Leitz filter A, cat. no. 513804), a FITC single band-pass filter (filter I3, cat. no. 513808), a TRITC single band-pass filter (filter N2.1, cat. no. 513812), a FITC/TRITC double band-pass filter (filter G/R, cat. no. 513803), and a triple filter (filter B/G/R, cat. no. 513836) for simultaneous observation of DAPI/Cascade-Blue, FITC and TRITC signals.

7. For image capturing, we use the software Metasystem Isis Version 5.0 (Metasystem, Altlusshein, Germany).

Was this article helpful?

0 0

Post a comment