In Vivo Systems

Either medium described in Section 2.3.3. can be used as a vehicle in which to transfer the embryos into the oviducts of rabbits or sheep and to flush the embryos out at the end of the culture period.

2.3.5.2. Cell-Free Systems

Ovine embryo culture from the one-cell to the hatched blastocyst stage utilizes SOFM+20% heat-inactivated human serum (HIHS) (14). Blood is collected from fasted males, the serum removed, heat-inactivated, and stored frozen in aliquots.

SOFM is made from five stock solutions as follows:

• Stock S: NaCl (31.47 g), KC1 (2.67 g), KH2P03 (0.81 g), Na lactate ([60%] 3.10 mL), glucose (1.35 g), penicillin (0.30 g), and streptomycin (0.25 g). Make to 500 mL with water, sterilize, store 3 mo.

• Stock B: NaHC03 (2.101 g) and phenol red (0.05 g). Make to 100 mL with water, sterilize, store 2 wk.

• Stock M: MgCl2-6H20 (0.500 g). Make to 50 mL with water, sterilize, store 3 mo.

• Stock C: Na pyruvate (0.072 g). Make to 20 mL with water, sterilize, store 1 wk.

• Stock D: CaCl2 • 2H20 (1.260g). Make to 50 mL with water, sterilize, store 3 mo.

Mix the stocks in the amounts listed below depending on volume needed, add HIHS to 20% (v/v), adjust pH to 7.4 and osmolarity to 280-300 mosM/L, and filter-sterilize. Discard after use.

Stock S

5.0 mL

or

1.0 mL

Stock B

5.0 mL

or

1.0 mL

Stock M

0.5 mL

or

0.1 mL

Stock C

0.5 mL

or

0.1 mL

Stock D

0.5 mL

or

0.1 mL

Water

38.5 mL

or

7.7 mL

50.0 mL

10.0 mL

It is also possible to use conditioned medium from oviduct cell cultures (see Section 2.3.5.3.).

2.3.5.3. Oviduct Cell Coculture

Secretions from oviduct cells are capable of supporting embryo development to the blastocyst stage (6,15,16). Although conditioned medium from oviduct cells can also be used, it is possibly easier to utilize the coculture technique. The media used contain standard items.

1. Earle's salts (Gibco Europe Ltd., Paisley, UK).

3. Newborn calf serum (NCS, Sera Lab. Ltd., Crawley Down, UK).

5. Kanamycin (Sigma).

6. 4-well culture plates (Nunc; Gibco).

2.4. Equipment

The production of transgenic sheep is costly in time, labor, and assets. All the equipment listed below is readily available. Much effort is being spent on methods to relieve this burden and methods with promise are examined in Section 1.

2.4.1. General Equipment

The production of media, embryo recovery and handling, embryo culture, and transgenic production requires that the laboratory be equipped to tissue-culture standard. Items required include:

1. pH meter and combination electrode (BJ321 Activon, Sydney, Australia).

2. Analytical balance

3. General glassware (top quality Pyrex) andplasticware (cell culture grade).

4. Tissue culture grade detergent (7X, ICN Biomedicals, Sydney, Australia).

5. An inverted microscope with a range of phase-contrast objectives (Nikon TMS, Nikon, Tokyo, Japan).

6. Binocular microscopes with magnification 2- to 65-fold for embryo searching.

7. An incubator for maintaining media and equipment at 38.5°C.

8. A laminar flow hood (DFMG4, Gelman Sciences Pty Ltd., Sydney, Australia).

9. Laparotomy cradle for restraint and elevation of sheep.

All glassware requires a thorough prewash with acid and extensive rinsing with tissue-culture grade water. Plasticware should be tested for suitability for embryo culture; mouse embryos can be used for this.

2.4.2. Intrauterine Insemination 2.4.2.1. Collection of Semen

This equipment is needed for collection and examination of sperm motility.

1. Artificial vagina (AV) and rubber lining.

2. Glass sperm-collecting vessel.

3. Dissecting microscope (Zeiss 47 50 57, Carl Zeiss, Oberkochen, Germany)

4. Cold light source (Schott KL 1500, Fibre Optics, Wiesbaden, Germany).

2.4.2.2. Insemination For intrauterine insemination, the following items are needed:

1. Full laparoscopic equipment including 7-mm diameter trocar-cannulae, a 6.5-mm 30° Hopkins laparoscope, grasping forceps, and high intensity fiber optic light source 485 (Karl Storz, Stennings, Sydney, Australia).

2. Carbogen cylinder and valve.

3. Insemination pipets, 150-mm long flame pulled and sharpened Pyrex glass tubing (4 mm od, 1 mm id). These may be reused after resharpening.

4. Adjustable cradle for restraint and support of ewes.

5. Osti animal clippers (Sunbeam Corp., Sydney, Australia).

6. Local anesthetic (2% procaine, Orgamol Talfar, Evionnaz, Switzerland).

7. Antibiotic (e.g., Penstrep, Troy Labs, Sydney, Australia).

8. Syringes and needles.

2.4.3. Embryo Recovery and Transfer

2.4.3.1. Surgical Equipment

1. Scalpel handle and blades, hemostats, scissors, suture, needle holder, suture needles, forceps, swabs, and towel clips.

2. Fenestrated drapes, 10-mL syringe, 18-g and 25-g needles, sterile normal saline.

3. Polyvinyl catheters 100-mm length (1.0 mm id x 1.5 mm od and 1.5 mm id x 2.0 mm od).

4. Induction anesthetic (Pentothal, Boehringer Ingelheim, Sydney, Australia).

5. Antibiotic (Penstrep).

6. Analgesic (Finadyne, Heriot Agvet Pty Ltd., Melbourne, Australia)

7. Topical antiseptic (Betadine, Faulding Pharmaceuticals, Adelaide, Australia).

8. Laparoscopic equipment (as described in Section 2.4.2.2.).

9. Sterile surgical tray.

2.4.3.2. Embryo Handling

1. Sterile embryo blocks and cover glasses.

2. Embryo handling pipets (flame pulled from Pasteur pipets to 250 |jmid).

3. Mouth tubes (Silastic tubing, 0.132 in. id x 0.183 in. od, Dow Corning, Midland, MI).

4. Depression slides for manipulation, thinnest ones selected for pronuclear visualization.

5. Drummond microdispenser (Drummond Scientific Co., Broomall, PA).

6. 3-1/2 French Tom-Cat catheter 5-1/2 in. long (Sherwood Medical, St. Louis, MO).

7. Jelco 16-g IV catheter placement unit (Johnson & Johnson, Tokyo, Japan).

2.4.4. Pronuclear Microinjection

2.4.4.1. Microscope and Optics

A research microscope with Nomarski accessories giving differential interference contrast (DIC) optics is essential.

1 Nikon Inverse Microscope Diaphot-TMD (Nikon, Japan). 2. Diascopic DIC Nomarski Attachment TMD-NT20.

2.4.4.2. Micromanipulators and Microinjector

For pronuclear microinjection, the following items are needed:

1. Two micromanipulators (e.g., Leitz micromanipulator, Ernst Leitz, Wetzlar, Germany).

2. Compressed air operated microinjector (Eppendorf 5242, Eppendorf, Carl Zeiss, Hamburg, Germany)

3. Micrometer syringe with luer fitting (Dixon Scientific Co., Beckenham, UK; Hamilton Co., Reno, NV).

The line connecting the syringe and the holding pipet is filled with fluorinert

2.4.4.3. Fine Needle Production

1. Capillary tubing (1.2-1.5 mm id, Kimble, Toledo, OH).

2. Glass tubing (GC 100TF.15, Clark Electromedical Instruments, Reading, UK).

3. Vertical Pipet Puller (Model 700D, David Kopf Instruments, Tujunga, CA).

4. Flaming Brown Micropipet Puller (Model P.80/PC, Sutter Instrument Co., San Rafael, CA).

Holding pipets are made from Kimble tubing and pulled on the Vertical Pipet Puller to 20-40 |iim id with the settings: heater 65 and solenoid 5. They are flame-polished before use. Microinjection needles are pulled from GC 100TF.15 glass tubing on the Flaming Brown using the settings: heat 400, pull 40, velocity 30, time 40. Settings on all pipet pullers will vary with environmental conditions.

2.4.5. Embryo Culture

1. IR 1500 Automatic C02 incubator (Flow Laboratories, Bioggio, Switzerland).

2. Sealed plastic container (e.g., a lunch box).

The box contains a stainless steel grid standing in, but extending above, a 1-cm layer of distilled water through which bubbles the gas mixture of 5% C02, 5% 02, and 90% N2 (Fig. 2).

Fig. 2. Diagram illustrating in vitro cell-free culture system. See Section 3.5.1.2.

2.4.6. Pregnancy Diagnosis

1. Ultrasound scanner (Vetscan, Tokyo Keiki, Tokyo, Japan).

2. Intracorporeal probe 5MHz linear transducer (Vetscan).

3. 3.5 MHz external linear transducer (Vetscan).

Other scanners and sector probes are also adequate for this work.

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