59 IN VITRO DEVELOPMENT OF FELINE CLONED EMBRYOS RECONSTRUCTED WITH PREADIPOCYTES

Abstract

The technique of somatic cell nuclear transfer (NT) in domestic cats is expected to contribute to the conservation of wildcats, for which extinction is a concern. In this study, we examined in vitro developmental ability of cloned embryos produced using the preadipocytes of domestic cats as nuclear donors. Primary cultures of preadipocytes were established as reported previously (Yagi et al. 2004 Biochem. Biophys. Res. Commun. 321, 967–974). Briefly, fat tissue (2–3 g) was excised from an adult female cat and digested using 0.1% collagenase for 1 h at 37°C followed by centrifugation. Only mature adipocytes that were floating near the surface of the supernatant were collected and placed in a 12.5-cm² culture flask filled with DMEM containing 20% FBS. The flask was filled with medium, tightly capped, and cultured upside down for 7–10 days, so that the floating adipocytes attached to the inner ceiling surface of the flask. When firm attachment of the cells to the ceiling surface of the flask was confirmed, the flask was then inverted and culture was continued using the routine cell culture technique for adherent cells. In vivo-matured oocytes were collected from the ovaries of domestic cats superovulated by eCG and hCG. IVM oocytes were obtained by culturing cumulus–oocyte complexes from the ovaries collected at local veterinary clinics in TCM199-based medium for 24 to 30 h. In vivo-matured and IVM oocytes were enucleated by aspirating the first polar body and adjacent cytoplasm using a bevelled pipette in the presence of 7.5 µg mL^–1 cytochalasin B. The nuclei of the donor cells were transferred to enucleated in vivo-matured and IVM oocytes by means of electric fusion (300 V mm^–1, 30 µs, twice). The reconstructed embryos were activated electrically (125 V mm^–1, 60 µs, twice), followed by treatment with 10 µg mL^–1 cycloheximide and 5 µg mL^–1 cytochalasin B. The cloned embryos were cultured in vitro for 7 days in MK-1 so that their developmental ability might be examined. The fusion rate of donor cells was similar between in vivo-matured and IVM oocytes (56.8%, 21/37 v. 54.5%, 42/77). The developmental ability of NT embryos reconstructed with in vivo-matured oocytes was similar to that of NT embryos reconstructed with IVM oocytes (cleavage: 52.4%, 11/21 v. 42.9%, 18/42; development to blastocysts: 9.5%, 2/21 v. 11.9%, 5/42). The results indicate that cloned feline embryos reconstructed using preadipocytes can develop in vitro into blastocysts.