69 PRODUCTION OF CLONED PIGS BY NUCLEAR TRANSFER OF PREADIPOCYTES

Abstract

Since the first success in producing cloned pigs, donor cells have been limited to fetal fibroblasts and a few other cell types. The aim of the present study was to determine if porcine preadipocytes can be efficient donor cells for somatic cell nuclear transfer (NT) in pigs. Preadipocytes established from subcutaneous adipose tissue of a male adult pig were used as nuclear donor cells. Cell cycle synchronization was carried out by serum starvation (5 days), confluency (5 days), roscovitine treatment (15 μM, 2 days), or differentiation induction by 0.5 mM 3-Isobutyl-1-methylxanthine, 0.25 μM dexamethasone, and 5 μg/mL insulin (5 days). Cell cycle synchronization and apoptosis of the donor cells were examined by flow cytometry and Annexin V staining and TUNEL. IVM oocytes were obtained from abattoir ovaries and matured in NCSU23. Donor cells were fused with the enucleated recipient oocytes by a single DC pulse of 200 V/mm for 10 μs in 0.28 M mannitol + 0.15 mM MgSO₄. Reconstructed embryos were electrically activated at 1–1.5 h after the NT, followed by cytochalasin B treatment for 3 h. Development of the NT embryos was assessed by fixation/staining at 3 h after NT, culture for 7 days in NCSU23, and transfer to the oviducts of estrus-synchronized recipient gilts. The cells immediately entered the G0 phase by differentiation induction (92.5 ± 0.4%), with higher efficiency of synchronization than for the other methods (roscovitine: 80.3 ± 0.2%; confluency: 79.9 ± 0.3%, P < 0.05) except for serum starvation (89.8 ± 0.6%). The proportion of apoptotic cells in the differentiation group was significantly lower than the other groups (Annexin V: 7.7% vs. 15.7 to 19.3%, TUNEL: 8.3% vs. 12.8 to 14.0%, P < 0.05). Incidence of premature chromosome condensation following NT (88.0%) was as high as that observed after NT with fetal fibroblasts previously (data not shown). In vitro developmental rates of the NT embryos did not differ significantly among the cell cycle synchronization methods of the donor cells (7.2 to 10.8%). Cell number of the blastocysts was highest in the differentiation group (49.0 vs. 30.2 to 41.9, P < 0.05). Transfer of 1004 cloned embryos of the serum starvation group to 5 recipients resulted in the production of 4 live and 1 stillborn piglets from 1 recipient. Transfer of cloned embryos reconstructed of donor cells treated by differentiation induction is currently underway. These data demonstrate that preadipocytes collected from an adult pig are promising nuclear donor cells for pig cloning.

This study was supported by PROBRAIN.