指導教授：吳信志臺灣大學：動物科學技術學研究所歐陽桓Ou-Yang, HuanHuanOu-Yang2014-11-282018-06-292014-11-282018-06-292014http://ntur.lib.ntu.edu.tw//handle/246246/262744小鼠Gzmg基因於小鼠胚胎二細胞期專一性的表現且是對於胚胎發育具有重要功能的絲胺酸分解酶。母源性胚源性基因轉變（Maternal Zygotic Transition；MZT）是胚胎發育中之第一個重要關卡：其特徵為母源性訊息的降解及胚源性基因的活化兩個過程，在小鼠中主要發生在第一次卵裂後的二細胞期。先前之研究結果顯示，Gzmg被抑制的小鼠胚胎會停滯於二細胞期，並且發現胚源性基因的活化出現障阻，顯示Gzmg在小鼠胚胎二細胞期對於MZT的發生是必要的。MZT的發生一直是目前研究較少的領域，因此了解Gzmg如何被專一性的表現於小鼠胚胎二細胞期，或許可提供MZT新的立基點。本研究首先擬從小鼠基因組中，完成選殖Gzmg之全長啟動子序列外，並擬完成選殖出包括Δ1- (-1369~+28nt), Δ2- (-939~+28nt), Δ3- (-711~+28nt) and Δ4-pGzmg (-417~+28nt)等，各不同長度片段的Gzmg啟動子序列。將pEGFP-N1載體中的CMV啟動子置換為不同片段的Gzmg啟動子。試驗首先使用顯微操作技術，共注射不同片段的Gzmg啟動子及作為背景值的pCMV-IRES2-mCherry載體於小鼠原核胚期及二細胞期的細胞核中，隨後處理Aphidicolin促使胚胎停滯於細胞週期的S期。再藉由JASPER進行轉錄因子結合位的預測，並輔以expression atlas晶片資料庫完成篩選可能存在於胚胎中的轉錄因子。最後，再以免疫螢光染色的方式，觀察該轉錄因子坐落於小鼠胚胎的時期及位置，並使用螢光標定的DNA與免疫螢光染色觀察是否具有交互作用。試驗結果證明在Δ4-pGzmg-EGFP組具有最高的轉錄活性，而FL- 及 Δ1-pGzmg-EGFP組則不僅轉錄活性較低，且於原核期胚其Δ4-pGzmg-EGFP之轉錄活性較顯著高於二細胞期胚胎。由以上結果，我們推論-417~+28nt的Gzmg啟動子序列含有時間專一性的促進子存在，並預測出Gabpa和Stat3可能是正調控Gzmg於二細胞期轉錄活性的轉錄因子。Gabpa及Stat3藉由免疫染色可以觀察到於小鼠胚胎一細胞期及二細胞期皆存在於胚胎中且坐落於細胞核中。在標定DNA結合免疫染色的方式，不論是Gabpa或Stat3皆可以觀察到與DNA有共坐落相同位置，此外Stat3會受到Δ4-pGzmg-EGFP的吸引而聚集於細胞核中。總和上述，Gzmg可能是Gabpa或Stat3於-417~+28nt的區域活化Gzmg的轉錄活性，藉由Gzmg活化後的功能促進胚胎的MZT的發生。Mouse Granzyme g (Gzmg) is an important protease and specificitly expressed at 2-cell stage of mouse embryo. Maternal zygotic transition (MZT) that control from maternal massage to synthesize zygotic gene products promote preimplantation embryo development in mouse 2-cell stage. It is demonstrated that zygotic RNA synthesize is blocked in Gzmg knock-down 2-cell embryo. These data indicated that Gzmg is a necessary protein in MZT. Thus, we want to know what mechanisms regulate Gzmg specific expression in mouse embryo 2-cell stage. We cloned full-length Gzmg promoter from mouse genomic DNA, FL-pGzmg (-1696 ~ +28nt), and generated four deletion constructs of Gzmg promoter, Δ1- (-1369 ~ +28nt), Δ2- (-939 ~ +28nt), Δ3- (-711 ~ +28nt) and Δ4-pGzmg (-417 ~ +28nt). We replaced CMV promoter of pEGFP-N1 with Gzmg promoter. Furthermore, we co-microinjected different types of Gzmg promoter constructs and pCMV-IRES2-mCherry vector into zygote pronuclear, 2-cell nuclear and also added an aphidicolin drug to arrest the embryo cell cycle in S phase. We predicted transcription factor binding sites in promoter sequence by JASPAR and expression atlas website. The immunofluorescence method can observe transcription factors location in mouse preimplantation embryo. We found the highest EGFP expression Δ4-pGzmg-EGFP, but lower expression level in FL and Δ1-pGzmg-EGFP in both zygote pronuclear and 2-cell nuclear injection. This result demonstrated that time-specific transcriptional factors up-regulated Gzmg transcription activity by binding cis-element in -417~+28nt of Gzmg promoter sequence. In addition, The JASER database and expression atlas website revealed that Gabpa and Stat3 were predicted that may up-regulate Gzmg in 2-cell stage. Futhermore, Gabpa and Stat3 located in zygote and 2-cell nuclear by immunofluorescence. These data supposed that Gabpa and Stat3 may had transcriptional activity of regulatory genes in zygote and 2-cell stage. Surprisingly, we observed that Gabpa and Stat3 co-localized with Δ4-pGzmg-EGFP. -939~+28nt of Gzmg promoter sequence had cis-element that controled Gzmg transcription activity, and Gabpa and Stat3 may up-regulate Gzmg in 2-cell stage. Thus, Gabpa and Stat3 are maternal proteins and may up-regulate Gzmg to promote MZT.誌謝………………………………………………………………………………………i 中文摘要………………………………………………………………………………...ii ABSTRACT………………………………………………………………………….....iii 目錄……………………………………………………………………………………...v 圖目錄…………………………………………………………………………………viii 表目錄……………………………………………………………………………...…...ix 壹、緒言...........................................................................................................................1 貳、文獻探討...................................................................................................................2 　一、著床前小鼠胚發育過程（Development program of preimplantation mouse embryo）…………………………….………………………………………......2 　　（一）著床前小鼠胚卵裂過程及初次分化…………………………...……........2 　　（二）著床前胚胎軸向及極性的建立...................................................................4 　　（三）早期胚胎基因表現趨勢...............................................................................6 　二、母源性與胚源性基因轉變（Maternal-Zygotic Transition；MZT）.................8 　　（一）母源性轉錄體及蛋白質對著床前胚胎發育之影響...................................8 　三、蛋白酶影響著床前小鼠胚發育.........................................................................11 　　（一）冷光-蛋白質複合體系統（Ubiquitin-Proteasome System；UPS）…….11 　　（二）Caspase及Granzyme的非細胞凋亡（apoptosis）途徑..........................13 　四、Granzyme 家族基因及其他非細胞凋亡機制....................................................15 五、基因轉殖動物產製方法.....................................................................................16 　　（一）原核注射……………………………………………………...……..……16 　　（二）反轉錄病毒載體法.....................................................................................16 　　（三）精子載體法（Sperm-mediated gene transfer；SMGT）..........................17 　　（四）胚胎幹細胞載體法………………………………………………..……...18 　　（五）體細胞核轉置法（Somatic cell nuclear transfer；SCNT）……….........18 　六、啟動子分析於著床前小鼠胚之方法.................................................................19 　七、研究動機及策略………………………………………………………..……...20 參、材料與方法.............................................................................................................21 　一、小鼠Gzmg啟動子表現載體之構築…………………………………………..21 　　（一）聚合酶連鎖反應（polymerase chain reaction；PCR）…………………21 　　（二）直接點突變法（site direct mutagenesis）.................................................24 　　（三）定序反應.....................................................................................................25 　　（四）酵素截切（Enzyme Digestion）................................................................26 　 （五）低溶點膠DNA回收法（Recovery of DNA from Low-Melting Temperature Agarose gels）………………..………………….........................26 　　（六）黏接作用（Ligation）……………………………………………..….….26 　　（七）轉型作用（Transformation）（EcosTM competent cell）........................27 　　（八）Cracking………………………………………………………………...…27 　　（九）菌落聚合酶連鎖反應（Colony PCR）……………………………….....27 　二、小鼠著床前胚胎啟動子分析.............................................................................28 　　（一）胚顯微注射用DNA之超高速離心純化………………………….…......28 　　（二）Plasmid Labeling Reaction（LabelITR TrackerTM Reagent）…….…....28 　　（三）小鼠胚胎收集.............................................................................................29 　　（四）原核與二細胞核顯微注射…………………………………………….....29 　　（五）螢光強度定量.............................................................................................30 　三、轉錄因子預測……………………………………………….…………………30 　　（一）JASPAR轉錄因子預測網頁…………………………..…………………30 　　（二）Expression Atlas基因晶片資料庫……………………..………………...30 　四、轉錄因子蛋白質位點檢測.................................................................................30 　　（一）全胚免疫螢光分析（whole-mount immune-fluorescence）………..…..30 　　（二）Cy5 標定DNA結合蛋白質免疫染色……………………………..…….31 肆、結果.........................................................................................................................32 　一、小鼠Gzmg啟動子表現載體之構築…………………………………………..32 　二、啟動子分析於小鼠著床前胚胎一至二細胞期.................................................33 　三、預測Gzmg啟動子序列可能含有之轉錄因子結合位.....................................39 　四、轉錄因子被觀察到在胚胎的細胞核中.............................................................43 　五、∆4-pGzmg-EGFP與轉錄因子的交互作用........................................................44 伍、討論.........................................................................................................................51 　一、在著床前小鼠胚胎的啟動子分析…………………………………………….51 　二、Stat3及Gabpa於小鼠胚胎發育的角色............................................................53 　三、總結.....................................................................................................................55 陸、結論.........................................................................................................................56 參考文獻.........................................................................................................................57 作者小撰……………………………………………………………………………….685047910 bytesapplication/pdf論文公開時間：2017/03/21論文使用權限：同意有償授權(權利金給回饋學校)Granzme g母源性胚源性基因轉變啟動子分析著床前胚胎thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/262744/1/ntu-103-R00626028-1.pdf