Germinal Vesicle Nuclear Transfer

2.1.1. Reagents

1. Pregnant mare serum gonadotropin (PMSG) (367222, Calbiochem).

2. Minimum essential medium (MEM) with Earle's salts and l-glutamine (11095080, Gibco, Invitrogen Corporation).

3. Cytochalasin D (C-8273, Sigma). Highly toxic.

5. Dimethylsulfoxide (DMSO) (D-2650, Sigma).

6. Light mineral oil (M-8410, Sigma).

2.1.2. Medium Preparation

1. In vitro maturation (IVM) medium for denuded mouse oocytes: MEM supplemented with 0.24 mM pyruvate (Pyruvic acid sodium salt, P-4562, Sigma) and 5 % fetal bovine serum (FBS; heat inactivated, F-7678, Sigma), 0.22-pm filter sterile. Pyruvate stock can be made at the concentration of 240 mM (1000X), aliquot, and stored at -20°C.

2. GV transfer micromanipulation medium: 25 mM HEPES (H-0763, Sigma)-buffered IVM medium, supplemented with 50 pg/mL 3-isobutyl-l-methylxan-thine (IBMX, I-7018, Sigma) and 2 pg/mL cytochalasin D, 0.22-pm filter sterile. IBMX stock can be made at the concentration of 50 mg/mL in DMSO (1000X), aliquot, and stored at -20°C. Cytochalasin D stock can be made at the concentration of 1 pg/pL in DMSO, aliquot, and stored at -20°C. (see Note 1).

3. Electro-fusion medium: 0.28 M mannitol (M-9647, Sigma), 0.1 mM CaCl2 (C-7902, Sigma), 0.1 mM MgSO4 (M-2393, Sigma), and 5 mM histidine (o2r 0.025% BSA) in deionized water, 0.22-pm filter sterile.

2.1.3. Equipment and Tools

1. Inverted microscope, equipped with Normarski interference optics, Axiovert 100TV, Zeiss (see Note 2).

2. Two micromanipulators, Narishige, Japan (see Note 3).

3. Two micro-syringes for applying suction or positive pressure, Narishige, Japan. Syringes, connecting plastic tubes, and holders are filled with Fluorinert FC-40 (Sigma), with no air bubbles in the path.

4. Microforge, MF-9, Narishige, Japan.

5. Pipet puller, Sutter P-97 (Sutter Instruments, Novato, CA).

6. Micro-grinder, EG-44, Narishige, Japan.

7. Borosilicate glass capillary tubing length 10 cm. Standard wall outside diameter 1.0 mm; inside diameter 0.58 mm. Thin wall outside diameter 1.0 mm; inside diameter 0.78 mm. Warner Instrument Corp.

8. Manipulation chamber, a simple chamber can be made by gluing a glass frame (using nontoxic glass glue) onto a long thin (25X55X1) glass coverslip, which allows maximum light passing through. Droplets (10-50 pL of manipulation medium with appropriate flatness to prevent light reflection) are made on the coverslip within the chamber covered with mineral oil to prevent evaporation.

9. Dissection stereomicroscope, SMZ-1B or 2B, Nikon, Japan.

10. CO2 incubator, Model 3130, Forma Scientific, Inc., USA.

11. BTX 2001 Electro Cell Manipulator®, Genetronics, Inc., San Diego, CA.

12. Alcohol burner.

13. Two pairs of Watchman forceps.

14. One pair of surgical forceps, and one pair of small curved forceps.

15. One pair of surgical scissors, and one small curved scissors.

16. Thirty-gauge needle with 1 mL disposable syringe.

17. Thirty-five-millimeter Petri dish (351008, Falcon).

18. Sixty-millimeter Petri dish (351007, Falcon).

19. Heat-pulled transfer pipets, with opening diameter of 100-120 pm.

2.1.4. Preparation of Microtools

1. Holding pipets. Holding pipets are made from capillaries (1 mm in diameter outside) above appropriately sized burner flame by turning around and pulling using two hands, to an outside diameter of approx 50-100 pm. A shaft of 1-1.5 cm is smoothly scratched using a diamond pen on clean Kimwipe supported by a finger, then bent slightly to create a clean flat cut. The tip is polished 45° over a glass bead melt on a platinum filament using a microforge, making inner diameter approx 15-20 pm. The pipette is reoriented and the shaft is bent about 10-15° with heated filament. The bent side is marked on the pipet for easy installation into the pipette holder (see Note 4).

2. Needles. Needles are produced from thick wall glass capillary tubing. The capillary tube is secured into the center of heating element in the pipet puller. The setting of the puller is selected to make the needle sharp at the tip and strong in the shaft. If this is not satisfied, the needle can be modified using the microforge.

3. Blunt nuclear transfer pipets. Transfer/enucleation pipettes are made from boro-silicate glass capillary tubing (1.0 mm outer diameter [OD], 0.78 mm inner diameter [ID]). The pipet is pulled on a horizontal pipet puller by adjusting appropriate heat, puller, velocity and time, so that a thin shaft of 15-30 pm less than 0.5 mm long is created. To make the instruments, we use Narishige microforge with a x10 objective, a platinum filament of diameter of 0.2 mm. The V-shaped filament is mounted horizontally in the microforge, and a small glass bead is fused onto the end of the filament. An even broken tip of an outside diameter of approx 15-20 pm is created by touching onto small glass bead made on a slightly heated thin platinum filament (0.1 or 0.2 mm), and instantly turning off heat using microforge. The heating on and rapid off constricts the glass and breaks the tip. The tip is slightly polished without visibly affecting opening of the tip, over a

Fig. 2. Germinal vesicle (GV) transfer by micromanipulation and fusion. (A-C) GV removal; (A) a GV oocyte sucked by a holding pipet; (B) Insertion of beveled pipet close to the GV; (C) The GV of donor oocytes is removed by a beveled pipet. Arrows indicate GV. (D-F) Reconstitution of GV oocytes; (D) A GV is placed in close contact with a recipient oocytes whose GV has been removed; (E) 30 min during fusion; (F) 1 h after fusion; GV is reconstituted with recipeint cytoplasm. Bar = 25 pm.

Fig. 2. Germinal vesicle (GV) transfer by micromanipulation and fusion. (A-C) GV removal; (A) a GV oocyte sucked by a holding pipet; (B) Insertion of beveled pipet close to the GV; (C) The GV of donor oocytes is removed by a beveled pipet. Arrows indicate GV. (D-F) Reconstitution of GV oocytes; (D) A GV is placed in close contact with a recipient oocytes whose GV has been removed; (E) 30 min during fusion; (F) 1 h after fusion; GV is reconstituted with recipeint cytoplasm. Bar = 25 pm.

glass bead melt on a platinum filament, to smooth the tip and avoid damage to eggs during micromanipulation.

4. Beveled nuclear transfer pipets. First, similar to making blunt end pipettes, an even broken tip of outside diameter approx 15-20 pm is made using a microforge after pipette pulling. A thin-walled capillary is drawn on the pipet puller at a setting that will produce a shaft of 10-15 mm from shoulder to tip. The pipet at an outside diameter of approx 20 pm is slightly fused to the glass bead near the tip of the filament, and the heat is switched off rapidly, such that contraction of the filament and glass break the tip off squarely and cleanly on the microforge, without causing narrowing of the tip. It is important not to bend the pipet when breaking on microforge. The pipet is then attached to a micro-grinder with high speed, lowered about 45° down onto grinding wheel, while sterile distilled water is being injected onto the wheel to clean the glass dust. The tip is beveled and clean (see Note 5 and Fig. 2).

The pipet tip must be sharpened for penetration of the zona pellucida using the microforge. The pipet is put back to the microforge, and the ground edge is then smoothed by bringing the bevel close to the glass bead, being heated a little. The tip of the pipet is touched to a small glass bead of fairly low heated filaments, such that the tip is a little fused with the bead, then the pipette is rapidly drawn away from the bead to form a short sharp spike. Care should be taken to avoid narrowing the opening. A long, sharp spike can cause damage to membrane and lysis of oocytes or embryos. So, it is also important not to stretch the tip when pulling the spike. Finally, the shaft of the pipet is bent about 10-15° on the side of the beveled tip with heated filament. This will ensure that the beveled tip can be seen under the microscope after mounting into the pipet holder, for convenience of micromanipulation (Fig. 2A). To avoid the occurrence of sticky cytoplasm, the pipet tip can be siliconized with Sigmacot (Sigma), washed with sterile distilled water, dried, and stored in a sealed container (see Note 6).

Pregnancy Guide

Pregnancy Guide

A Beginner's Guide to Healthy Pregnancy. If you suspect, or know, that you are pregnant, we ho pe you have already visited your doctor. Presuming that you have confirmed your suspicions and that this is your first child, or that you wish to take better care of yourself d uring pregnancy than you did during your other pregnancies; you have come to the right place.

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