学会発表Trial of nuclear transfer from bovine freeze-dried fibroblast cells
Freeze-dried (FD) somatic cells have been proposed as a new tool for producing cloned animals, since it could overcome disadvantages of the current cryopreservation method. Recently it was succeeded to develop into blastocysts by nuclear transfer (NT) using FD somatic cells in the sheep and pig. Furthermore, chimeric mice were produced by ES cell lines from nuclear transferred blastocysts of FD cells. We investigated the influence of freeze drying solution on DNA damage of bovine fibroblast cells after lyophilization, and the ability of in vitro development of oocytes after NT. In addition, OCT4 and IFN-tau expression of NT and IVF embryos were examined. Bovine fibroblast cells were obtained from ear skin tissues of a Japanese Brown Cattle-Kochi. The cells were suspended in m-EGTA (50 mM EGTA, 100 mM Tris HCl, pH 8.2) and Na-EGTA (50 mM NaCl, 50 mM EGTA, 10 mM Tris HCl, pH 8.2), and lyophilized for 11.5 h. The DNA damage of the FD cells was evaluated by alkaline comet assay. The rehydrated cells were injected into enucleated bovine oocytes. Activation treatment was accomplished by ionophore A23187 and 6-dimethylaminopyridine. NT embryos were cultured in CR1aa medium for 7 days. The DNA-damaged cells were significantly fewer in m-EGTA than in Na-EGTA (12% and 24%, p < 0.05). The cleavage rates of NT embryos in m-EGTA and Na-EGTA were 50% and 56%, and the blastocyst formation rates were 10% and 0%, respectively. No significant difference in OCT4 expression was observed between NT and IVF embryos. Whereas IFN-tau expression of NT embryos was lower than that of IVF embryos. These results suggest that m-EGTA is better than Na-EGTA for a freeze drying solution. Further studies are necessary in order to produce bovine clones from FD cells.