Cloning Formosan Black Bear embryos using recipient ooplasms of porcine and caprine

自桃麗複製羊(Dolly the Sheep)出生後，體細胞核轉置(somatic cell nuclear transfer, SCNT)已被普遍應用於複製不同之家畜與野生動物。本試驗之目的為評估以豬與山羊卵母細胞為受核源複製台灣黑熊(Ursus thibetanus formosanus)種間重組胚之效率。試驗一探討血清饑餓處理時間與細胞生長密度(confluency)對台灣黑熊耳朵纖維母細胞細胞週期分佈之影響。經流式細胞儀(flowcytometer)分析顯示，台灣黑熊耳朵纖維母細胞分別培養至30%、60%及90%之細胞生長密度後發現，其細胞週期分佈於G0/G1期者(79.53-87.33%)並無顯著差異(P > 0.05)。而經血清饑餓處理1-6天(79.8-84.0% vs. 78.0%, P > 0.05)或6-10天(78.7-83.3% vs. 74.3%)之台灣黑熊耳朵纖維母細胞分佈於G0/G1期之百分率亦與對照組者相似(P > 0.05)。試驗二，以豬卵母細胞質接受台灣黑熊耳朵纖維母細胞複製種間重組胚，並在培養液中添加組蛋白去乙醯化酶抑制劑Trichostatin A (TSA)，探討其對台灣黑熊-豬複製胚體外發育之影響。體外成熟之豬卵母細胞接受經血清饑餓(serum starvation)之台灣黑熊耳朵纖維母細胞後，複製胚經孤雌激活並培養於含25 nM或50 nMTSA 之Porcine Zygote Medium-3 (PZM-3)中一天，再轉培養於不含TSA之PZM-3中直到第六天。結果顯示，組蛋白乙醯化強度雖然於對照組與TSA處理組之複製胚有所增加，但多數複製胚無法發育至4-細胞期以上。試驗三，以山羊卵母細胞為受核卵質，接受台灣黑熊耳朵纖維母細胞後培養於TCM-199中7天，其中僅有一複製胚可發育達18-細胞(0.8%, n=115)，其餘胚皆停滯於8-細胞期前(90.6%)。最終，若以豬卵質複製之台灣黑熊重組胚注入台灣黑熊粒線體後，其分裂率顯著低於未注入粒線體之對照組(34% vs. 68%, P < 0.05)，大部分之複製胚停滯於1-細胞期(29%)，僅有少數(6%)可發育至2-或3-細胞期。 綜合上述研究結果發現，台灣黑熊耳朵纖維母細胞經血清饑餓延長至10天並未增加其G0/G1期之比例。TSA與粒線體注射仍無法有效改善台灣黑熊複製胚之發育潛能。因此，異種間複製胚發育之調控機制仍有待更多的研究與瞭解。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.文獻檢討--------------------------------------------------------------------------------------------1 前言--------------------------------------------------------------------------------------------------2 一、 改善SCNT與iSCNT效率之因子---------------------------------------------------------3 (一) 受核卵質與供核細胞之細胞週期---------------------------------------------------3 (二) 供核細胞與受核卵質間粒線體(mitochondrion)之交互作用--------------------5 二、 組蛋白去乙醯化酶抑製劑(histone deacetylase inhibitor)對複製胚組蛋白乙醯化(acetylation)之影響----------------------------------------------------------------6 (一) Trichostatin A (TSA)----------------------------------- ---------------------------------7 (二) 丁酸鈉(sodium butyrate, NaBu) -----------------------------------------------------8 (三) Scriptaid-----------------------------------------------------------------------------------8 三、 iSCNT胚之基因與蛋白質表現異常----------------------------------------------------12 研究試驗-------------------------------------------------------------------------------------------14 試驗一、 台灣黑熊耳朵纖維母細胞經血清肌餓處理後之細胞週期分佈-------------15 一、 前言---------------------------------------------------------------------------------------15 二、 材料與方法------------------------------------------------------------------------------15 三、 試驗設計---------------------------------------------------------------------------------17 四、 統計分析---------------------------------------------------------------------------------18 五、 結果---------------------------------------------------------------------------------------18 六、 討論---------------------------------------------------------------------------------------23 試驗二、 於胚培養液中添加Trichostatin A (TSA)對以豬卵母細胞產製之台灣黑熊種間複製胚體外發育之影響-------------------------------------------------------------------25 一、 前言---------------------------------------------------------------------------------------25 二、 材料與方法------------------------------------------------------------------------------25 三、 試驗設計---------------------------------------------------------------------------------34 四、 統計分析---------------------------------------------------------------------------------34 五、 結果---------------------------------------------------------------------------------------34 六、 討論---------------------------------------------------------------------------------------39 試驗三、 以山羊卵母細胞產製台灣黑熊種間複製胚與注射粒線體對其體外發育能力之影響-------------------------------------------------------------------------------------------41 一、 前言---------------------------------------------------------------------------------------41 二、 材料與方法------------------------------------------------------------------------------41 三、 試驗設計---------------------------------------------------------------------------------46 四、 統計分析---------------------------------------------------------------------------------47 五、 結果---------------------------------------------------------------------------------------47 六、 討論---------------------------------------------------------------------------------------52 結論-------------------------------------------------------------------------------------------------53 附錄一、 培養液中添加TSA對迷你豬複製胚體外發育之影響-------------------------------------------------------------------------------------------------------54 一、 材料與方法------------------------------------------------------------------------------54 二、 試驗設計---------------------------------------------------------------------------------57 三、 統計分析---------------------------------------------------------------------------------57 四、 結果---------------------------------------------------------------------------------------57 參考文獻-------------------------------------------------------------------------------------------6