23 PRODUCTION OF HANDMADE CLONED GOAT BLASTOCYSTS USING FETAL FIBROBLAST CELLS

Abstract

Nuclear transfer is a very effective method for propagation of desired, extinct, and endangered animals as well as for the production of 100% transgenic animals. Enucleated oocytes and somatic cells are required for nuclear cloning. For enucleation, DNA-specific stains are used for visualization of the metaphase (MII) plate in matured oocytes under UV illumination in both micromanipulator-based and handmade cloning techniques. The present study was carried out to produce cloned goat embryos using the handmade cloning approach. Fetal fibroblast cells were used as nuclear donors (passages 3–4). Oocytes were collected from slaughterhouse-derived ovaries and matured in maturation medium (TCM-199 (HEPES modified), 5 µg mL^–1 FSH, 10 µg mL^–1 LH, 1 µg mL^–1 estradiol-17β, 50 µg mL^–1 gentamicin, 3 mg mL^–1 BSA, and 10% inactivated estrus goat serum) at 38.5°C in 5% CO₂ in air with maximum humidity for 24 h. We observed that the formation of transparent protrusion cones on the surface of the in vitro-matured goat oocytes was clearly visible under the stereomicroscope after zona digestion with 2 mg mL^–1 pronase. The extent of protrusion cone formation in matured oocytes was 95–100% within 20–30 min in handling medium T 20 (TCM-199 + 20% FCS). The MII plate in the protrusion cone was confirmed (100%) after Hoechst 33342 staining and subsequent UV illumination under the inverted microscope. Zona-free oocytes were bisected on the basis of the protrusion cone by a microblade in medium (T 20 + 2.5 µg mL^–1 cytochalasin B) for enucleation. Enucleated demi-oocytes were selected which had no protrusion cone and were without staining. Fetal fibroblasts from confluent monolayers were used. Two demi-oocytes were coupled with one trypsinized fetal fibroblast cell using 200 µg mL^–1 phytohemagglutinin. The triplets were fused together with a combination of alternating current (7 V) and direct current (2.31 kV cm^–1 for 15 µs with a double pulse) in fusion medium (0.3 m mannitol, 0.1 mM MgSO₄, 0.05 mm CaCl₂, and 3 mg mL^–1 BSA). Four h after fusion, reconstructed oocytes were activated by using 2 µm Ca Ionophore for 5 min at room temperature and incubated with 2 mm 6-dimethylaminopurine at 38.5°C in 5% CO₂ in air for 3 h. Activated reconstructed embryos were cultured in embryo development medium (TCM-199, 10% FCS, essential and nonessential amino acids, and 10 mg mL^–1 BSA) in the well of the well (WOW) culture system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 258–264) at 38.5°C in 5% CO₂ in air. In the present study, fusion, cleavage, and morula and blastocyst formation rates were 180/200 (90%), 72/180 (40%), 56/72 (77%), and 6/56 (11%), respectively. Further studies will be required to optimize blastocyst production. In conclusion, the protrusion cone formation in matured goat oocytes made it convenient for bisection and enucleation without Hoechst staining and UV light exposure, enabling the production of goats from handmade somatic cell cloning.

The Council of Scientific and Industrial Research, India, has provided a fellowship to the first author to carry out this research work.