| Summary: | 碩士 === 國立中興大學 === 動物科學系所 === 100 === After the birth of Dolly the Sheep, somatic cell nuclear transfer (SCNT) has been successfully applied to clone a wide variety of livestock and wild species. In this study, we aimed to evaluate the efficiency of producing interspecies Formosan Black Bear (Ursus thibetanus formosanus, FBB) embryos using pig and goat oocytes. In Experiment 1, flow cytometry was used to determine the influence of cell confluency and serum starvation on the distribution of the FBB ear fibroblasts in several cycle phases (G0/G1, S, and G2/M). The results showed that FBB ear fibroblasts populated in G0/G1 phase were not significantly different from those of the control group (79.8-84.0% vs. 78.0%, and 78.7-83.3% vs. 74.3%, P > 0.05) after serum starvation for 1 to 10 days, neither among those cultured with 30-90% cell confluency (79.5-87.3%, P > 0.05). In Experiment 2, the effect of Trichostatin A (TSA), a histone deacetylase inhibitor, on the development of reconstructed FBB embryos was tested. Reconstructed embryos after parthenogenetic activation were cultured in Porcine Zygote Medium-3 (PZM-3) supplemented with 25 nM or 50 nM of TSA for one day and then switched to PZM-3 without TSA for six more days. Developmental competence was assessed and the results showed that cloned FBB-porcine embryos could not develop beyond the 4-cell stage after in vitro culture in the control and the TSA-treated groups, although the intensity of histone acetylation was enhanced in the TSA-treated groups. In Experiment 3, FBB interspecies embryos were cloned by using goat oocytes receiving donor cells from FBBs. The reconstructed embryos were cultured in TCM-199, but only one cloned embryo reached the 18-cell stage (0.8%, n=115) and the majority (90.6%) of cloned embryos arrested before the 8-cell stage. In a separate study, FBB interspecies embryos cloned by using porcine oocytes injected with FBB mitochondria were cultured in the PZM-3 medium. The results showed that cloned FBB-porcine embryos had a lower cleavage rate than the uninjected group (34% v. s 68%, P < 0.05). Only some of the cloned FBB-porcine embryos with mitochondria injection reached the 2- (29%) or 3-cell stage (6%), but the majority of them remained uncleaved.
In conclusion, cultured FBB fibroblasts exhibit a high proportion of cells resting in the G0/G1 phase, and serum starvation treatment does not increase their G0/G1 cell population. Both TSA treatment and mitochondrion injection could not enhance the development of cloned FBB embryos beyond the cleavage stage, suggesting more complicate mechanisms involved in cloning the interspecies embryos for conservation.
|
|---|
