- 學位論文
種間體細胞核轉置產製石虎複製胚之探討
Cloning of the Leopard Cat Embryos by Interspecies Somatic Cell Nuclear Transfer
摘要
臺灣石虎（Prionailurus bengalensis）是臺灣唯一現存的野生貓科動物， 2017年被臺灣陸域哺乳類動物紅皮書名錄列為國家瀕危物種 (nationally endangered, NEN)。體細胞核轉置（somatic cell nuclear transfer, SCNT）技術已應用於超過20種以上的哺乳動物並成功產下複製個體，故被視為延續瀕危物種的措施之一。本研究將以臺灣石虎成纖維母細胞為供核細胞，並以體外成熟的家貓卵母細胞經去核後作為受核細胞，其後透過電融合與激活處理產製種間體細胞核轉置(interspecies SCNT, iSCNT)胚。試驗的第一階段係自獸醫院採集結紮手術切除的家貓卵巢，並從0.5-1.0 mm的腔狀濾泡中收集未成熟卵母細胞，置於M199 + 0.4% BSA培養液中進行體外培養24 h，建立家貓卵母細胞體外成熟（in vitro maturation, IVM）系統之建置；此外，本試驗亦評估卵巢4 oC冷藏保存與家貓個體年齡對體外成熟的影響。經24 h冷藏保存的卵巢，相較於新鮮採集者其卵母細胞體外成熟率並無顯著差異(73.9%, 147/199 vs. 68.8%, 130/189; p > 0.05)。而卵母細胞源自< 1 歲、1-2歲與> 2 歲之母貓其成熟率分別為68.3% (86/126)、56.5% (35/62)及59.4% (41/69) (p < 0.05)。結合電刺激(1 kV/cm, 20 μsec, 2 pulses)與6-DMAP (2 mM, 3 h)處理，可使家貓卵母細胞成功孤雌激活並發育至囊胚(24.2%, 12/61)，囊胚細胞數可達128±13，並具有結構緻密完整之內細胞團(inner cell mass, ICM)。進一步經螢光免疫染色分析，結果顯示家貓早期胚於16-細胞期開始表現胚源性的SOX2與OCT4，而CDX2則於第8天的擴張囊胚才開始表現，直到第9天的孵化囊胚，SOX2與OCT4訊號轉集中於ICM區域，而CDX2則僅表現於滋養層細胞。在供核細胞方面，家貓和石虎成體成纖維母細胞經0.5%血清飢餓處理3天後，86.3% 及79.9% (n=3)成纖維母細胞之細胞週期可同期化至G1/G0期；以1 μM Trichostatin A (TSA, HDAC抑制劑)處理24 h後，H3K18免疫染色顯示與未處理組比較即呈顯著整體乙醯化之趨勢；經TSA處理後之成纖維母細胞其核轉置胚(相較於未處理組)之囊胚率(17.6%, 10/64 vs. 3.5%, 2/57, p < 0.05) 、囊胚細胞數(108±22 vs. 94±14)，以及各胚期之H3K9乙醯化表現皆顯著較高於未經TSA處理組(p < 0.05)，並皆正常表現SOX2與OCT4。本試驗最後成功產製石虎iSCNT複製囊胚(10.2％, 6/98)，細胞數亦可達114±31，並具正常的H3K9乙醯化與OCT4表現，此為複製石虎胚成功再程序化的表徵。後續透過微衛星DNA分析證實iSCNT胚確實源自石虎成纖維母細胞，而粒線體DNA (mtDNA)分析亦指出iSCNT複製胚的胚源性基因經活化後，可觀察到源自供核石虎體細胞之mtDNA呈再活化之趨勢。綜上所述，本研究證實經體外成熟的家貓卵母細胞質具有再程序化石虎體細胞的潛能，進而成功產製異種核移殖之臺灣石虎複製囊胚。研究結果可提供野生貓科瀕危物種資源的保存之參考。
並列摘要
Leopard cat (Prionailurus bengalensis), the only wild feline still inhabiting the main island of Taiwan, has been listed as Nationally Endangered (NEN) in The Red List of Terrestrial Mammals of Taiwan since 2017. Somatic cell nuclear transfer (SCNT) has been applied for more than 20 species and is considered a reliable tool for preserving the genetics of endangered species. Here, we propose to produce cloned leopard cat embryos by interspecies SCNT (iSCNT) using fibroblast cells derived from leopard cats with enucleated oocytes from domestic cats. Firstly, the in vitro maturation (IVM) system for domestic cat oocytes was established. The immature oocytes were collected from ovariectomy and subsequently in vitro culture for 24 h in the maturation medium containing M199 + 0.4% BSA till the first polar body extrusion. The effects on the maturation rate of ovary cold storage and donor age were also assessed. The MII maturation rate derived from ovaries stored at 4oC for 24 h showed no difference with fresh collected (73.9%, 147/199 vs. 68.8%, 130/189), The maturation rate of oocytes collected from donors below 1-year-old, from 1-2 years old, and over 2-year old were 68.3% (86/126), 56.5% (35/62) and 59.4% (41/69), respectively. The parthenogenetic activation of oocytes was conducted by electroporation (1 kV/cm, 20 μsec, 2 pulses) combined with the treatments of 2 mM 6-DMAP for 3h. The blastocyst rate was 24.2% (12/61) with cell number 128±13, also showing the compact structure of inner cell mass (ICM). Furthermore, the expression patterns of SOX2, OCT4, and CDX2 in domestic cat preimplantation embryos were first illustrated with the immunofluorescence staining. The expressions of zygotic SOX2 and OCT4 were observed from the 16-cell stage, while CDX2 was detected in Day-7 early blastocysts and later to be evenly expressed in Day-8 expanded blastocysts. It is noteworthy that in Day-9 hatching blastocysts, both SOX2 and OCT4 expression were concentrated toward the ICM region, and CDX2 was strictly expressed only in trophectoderm (TE) cells. Next, both domestic cat and leopard cat adult fibroblasts were used as donor cells for SCNT. With 0.5% serum starvation for 3 days and the treatment of 1 μM TSA for 24h, 86.3% and 79.9% (n=3) of the domestic cat and leopard cat fibroblast cells were synchronized at G1/G0 phase and achieved global acetylation confirmed by immunofluorescent staining of AcH3K18. Following SCNT, a significant improvement in blastocyst rate and cell number was achieved with serum starvation plus pretreatment of TSA (17.6%, 108±22), compared to those from the control group (3.5%, 2/57, 94±14). The AcH3K9 level among developmental stages was also found significantly higher in TSA treated group (p < 0.05). Moreover, the iSCNT cloned embryos were generated by the fusion of leopard cat nuclei with domestic cat cytoplasts, with compatible blastocyst rate (10.2%, 6/98) and cell number (114±31). The microsatellite analysis confirmed the nuclear genome of iSCNT cloned embryos was from leopard cat origin, and the mtDNA analysis showed possible regaining of leopard cat mtDNA in iSCNT cloned embryos following gene activation. Collectively, this present study demonstrated that the properties of domestic cat oocytes could be used to reprogram the genomes of leopard cats in Taiwan and successfully develop to the blastocyst stage. Our data provided a possible solution for endangered species conservation, particularly the Felidae family.