Abstract
The aim of the present study was to investigate whether a somatic stem cell derived from the salivary gland can be an efficient donor cell for pig cloning. Somatic stem cells (salivary gland progenitor cells, SGP) were isolated from the salivary gland of a 4-month old male pig (Matsumoto et al. 2004). Briefly, tissue sections of salivary gland were gently digested by collagenase/hyaluronidase and dispersed into single cells. Isolated cells (5 × 104) were cultured on a type I collagen-coated dish in William's medium E; then colonies having epithelial-like morphology were picked to establish the primary culture of SGP cells (CD49, intracellular laminin-positive) which have the potential to differentiate in vitro into hepatic and pancreatic endocrine cells after spherical body formation in vitro. SGP cells to be used as nuclear donors were cultured for 2 days under serum starvation. Fetal fibroblast (FF) cells were used as control nuclear donors. 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 190 V/mm for 10 μs in 0.28 M mannitol + 0.15 mM MgSO4. Reconstructed embryos were electrically activated by DC pulse of 150 V/mm for 100 μs in 0.28 M mannitol + 0.05 mM CaCl2 + 0.1 mM MgSO4 at 1–1.5 h after the NT, followed by cytochalasin B treatment for 3 h. Development of the NT embryos was assessed in vitro by fixing and staining at either 2 h post NT or after culture for 7 days in NCSU23, or in vivo by transfer to the oviducts of estrus-synchronized recipient gilts. Incidence of premature chromosome condensation was similar regardless of donor cell type. Development of the NT embryos reconstructed with SGP to the blastocyst stage was significantly higher compared to that of the FF group (38/137, 27.7% vs. 19/168, 11.3%, respectively; P < 0.05). Transfer of 278 cloned embryos reconstructed with SGP to two recipients resulted in the production of three live piglets. Production efficiency of piglets from the cloned embryos reconstructed with FF was 2/263. Based on the in vitro development of the reconstructed embryos, SGP is a promising nuclear donor cell for pig cloning; further transfer experiments are to be carried out. This study was supported by PROBRAIN.
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