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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 李士傑(Shyh-Jye Lee) | |
dc.contributor.author | Ssu-Ting Lin | en |
dc.contributor.author | 林思婷 | zh_TW |
dc.date.accessioned | 2021-06-16T03:44:24Z | - |
dc.date.available | 2020-03-13 | |
dc.date.copyright | 2015-03-13 | |
dc.date.issued | 2015 | |
dc.date.submitted | 2015-02-09 | |
dc.identifier.citation | Amanze D, Iyengar A. 1990. The micropyle: a sperm guidance system in teleost fertilization. Development 109: 495-500
Burkel BM, von Dassow G, Bement WM. 2007. Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophin. Cell motility and the cytoskeleton 64: 822-32 Chan D, Thomas CJ, Taylor VJ, Burke RD. 2013. Integrins on eggs: focal adhesion kinase is activated at fertilization, forms a complex with integrins, and is necessary for cortex formation and cell cycle initiation. Molecular biology of the cell 24: 3472-81 Cheng JC, Miller AL, Webb SE. 2004. Organization and function of microfilaments during late epiboly in zebrafish embryos. Developmental dynamics : an official publication of the American Association of Anatomists 231: 313-23 Chun JT, Limatola N, Vasilev F, Santella L. 2014. Early events of fertilization in sea urchin eggs are sensitive to actin-binding organic molecules. Biochemical and biophysical research communications Chun JT, Puppo A, Vasilev F, Gragnaniello G, Garante E, Santella L. 2010. The biphasic increase of PIP2 in the fertilized eggs of starfish: new roles in actin polymerization and Ca2+ signaling. PloS one 5: e14100 Chun JT, Santella L. 2009. The actin cytoskeleton in meiotic maturation and fertilization of starfish eggs. Biochemical and biophysical research communications 384: 141-3 Chun JT, Vasilev F, Santella L. 2013. Antibody against the actin-binding protein depactin attenuates Ca2+ signaling in starfish eggs. Biochemical and biophysical research communications 441: 301-7 dos Remedios CG, Chhabra D, Kekic M, Dedova IV, Tsubakihara M, et al. 2003. Actin binding proteins: regulation of cytoskeletal microfilaments. Physiological reviews 83: 433-73 Hart NH, Becker KA, Wolenski JS. 1992. The sperm entry site during fertilization of the zebrafish egg: localization of actin. Molecular reproduction and development 32: 217-28 Jang Y, Soekmadji C, Mitchell JM, Thomas WG, Thorn P. 2012. Real-time measurement of F-actin remodelling during exocytosis using Lifeact-EGFP transgenic animals. PloS one 7: e39815 Joseph N, Reicher B, Barda-Saad M. 2014. The calcium feedback loop and T cell activation: how cytoskeleton networks control intracellular calcium flux. Biochimica et biophysica acta 1838: 557-68 Kinsey WH. 2009. Analysis of signaling pathways in zebrafish development by microinjection. Methods in molecular biology 518: 67-76 Leu DH, Draper BW. 2010. The ziwi promoter drives germline-specific gene expression in zebrafish. Developmental dynamics : an official publication of the American Association of Anatomists 239: 2714-21 Munsie LN, Caron N, Desmond CR, Truant R. 2009. Lifeact cannot visualize some forms of stress-induced twisted F-actin. Nature methods 6: 317 Otto JJ, Kane RE, Bryan J. 1980. Redistribution of actin and fascin in sea urchin eggs after fertilization. Cell motility 1: 31-40 Puppo A, Chun JT, Gragnaniello G, Garante E, Santella L. 2008. Alteration of the cortical actin cytoskeleton deregulates Ca2+ signaling, monospermic fertilization, and sperm entry. PloS one 3: e3588 Reddy P, Deguchi M, Cheng Y, Hsueh AJ. 2013. Actin cytoskeleton regulates Hippo signaling. PloS one 8: e73763 Riedl J, Crevenna AH, Kessenbrock K, Yu JH, Neukirchen D, et al. 2008. Lifeact: a versatile marker to visualize F-actin. Nature methods 5: 605-7 Sampath P, Pollard TD. 1991. Effects of cytochalasin, phalloidin, and pH on the elongation of actin filaments. Biochemistry 30: 1973-80 Santella L, Chun JT. 2011. Actin, more than just a housekeeping protein at the scene of fertilization. Science China. Life sciences 54: 733-43 Tsaadon A, Eliyahu E, Shtraizent N, Shalgi R. 2006. When a sperm meets an egg: block to polyspermy. Molecular and cellular endocrinology 252: 107-14 Webb SE, Miller AL. 2013. Ca(2+) signaling during activation and fertilization in the eggs of teleost fish. Cell calcium 53: 24-31 Winder SJ, Ayscough KR. 2005. Actin-binding proteins. Journal of cell science 118: 651-4 Wolenski JS, Hart NH. 1987. Scanning electron microscope studies of sperm incorporation into the zebrafish (Brachydanio) egg. The Journal of experimental zoology 243: 259-73 Wuhr M, Obholzer ND, Megason SG, Detrich HW, 3rd, Mitchison TJ. 2011. Live imaging of the cytoskeleton in early cleavage-stage zebrafish embryos. Methods in cell biology 101: 1-18 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55015 | - |
dc.description.abstract | 肌動蛋白(Actin)是真核生物含量最高的蛋白質之一,也是細胞骨架的必要元素之一。肌動蛋白絲(F-actin)由肌動蛋白單元(G-actin)構成,參與很多重要的生理功能,包含細胞形狀的形成及維持、細胞移動、細胞內物質運輸和細胞分裂,所以發展監測肌動蛋白絲變化的工具對其功能的研究相當重要。受精作用從精子接觸到卵子開始,過程中會伴隨著一系列劇烈的變化,最後發育成一全新的生命個體。在硬骨魚的卵表面上有一個由肌動蛋白絲構成的漏斗狀結構稱為卵膜孔(micropyle),精子會從此處進入魚卵中。而在海膽及老鼠的研究中已經證實肌動蛋白絲於受精之後會立刻進行重組,更有研究指出,肌動蛋白絲的調控跟受精後會出現的鈣離子波動有關。目前在斑馬魚研究中缺乏適合監測肌動蛋白絲的工具,為了研究肌動蛋白絲在受精及胚胎發育時期所扮演的角色,我們發展了一個方法來監測斑馬魚卵中的肌動蛋白絲變化。我們在斑馬魚卵中利用ziwi促進子驅動與綠螢光蛋白融合的utrophin (GFP-UtrCH)的表現。在雌性斑馬魚中,ziwi促進子可以在所有分化階段的卵細胞(包含成熟卵)中驅動基因表現,而GFP-UtrCH是一個可以做為肌動蛋白絲探針的重組蛋白。經由與肌動蛋白絲劑rhodamine phalloidin染色結果的比對,我確認在ziwi:GFP-UtrCH 基因轉殖魚卵和胚胎中的螢光訊號可以即時反映肌動蛋白絲的位置及變化。接著我用ziwi:GFP-UtrCH 基因轉殖魚卵實行體外受精並可以同時監測到卵中肌動蛋白絲的變化。此基因轉殖斑馬魚未來將可作為研究肌動蛋白絲在受精及胚胎發育時期變化的有力工具。 | zh_TW |
dc.description.abstract | Actin is one of the most abundant proteins in eukaryote and is an essential component of cytoskeleton. Filamentous actin (F-actin), assembled by polymerization of globular actin (G-actin) monomers, is involved in many crucial physiological functions, such as the formation and maintenance of cell shape, cell motility, cell division and intracellular trafficking. Consequently, the development of tools for monitoring F-actin dynamic is important to understand the functional roles of actin during development and other physiological processes. Fertilization starts with sperm contacting eggs, and followed by dramatic changes inside egg to form a complex muticellular organism. In teleost, there is a specific sperm entry site called micropyle, a dimple-like structure consisting of circular tuft of microvilli and containing a meshwork of actin filaments. Furthermore, the F-actin network of unfertilized egg rearranges immediately to the egg cortex during/after fertilization in species such as sea urchin and mouse. Some studies also indicate that the fine regulation of actin filament may be related to calcium wave trigger by fertilization.
I was interested in studying actin dynamic during fertilization and early embryogenesis. Due to the lack of a suitable F-actin reporting line in zebrafish, I used a ziwi promoter to drive the maternal expression of green fluorescent protein-utrophin (GFP-UtrCH) fusion proteins. The ziwi promoter is a germ cell-specific promoter and GFP-UtrCH is an F-actin probe, which can bind F-actin by its calponin homology domain. In various development stages of Tg(ziwi:GFP-UtrCH) embryos, the fluorescent protein signal faithfully represent the distribution of F-actin that resembles the rhodamine phalloidin staining patterns. I also performed in vitro fertilization using the unfertilized egg from Tg(ziwi:GFP-UtrCH) and observed dramatic reorganization of cortical actin. This transgenic line is thus an effective tool to study the regulation of F-actin dynamics during fertilization and subsequent development. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T03:44:24Z (GMT). No. of bitstreams: 1 ntu-104-R01b41016-1.pdf: 1580543 bytes, checksum: 626524c3595efcfc3610efc2400f31ec (MD5) Previous issue date: 2015 | en |
dc.description.tableofcontents | 口試委員會審定書 I
誌謝 II 中文摘要 III Abstract IV Content VI List of Figures VIII Introduction 1 Actin and Actin Dynamic 1 The Changes of the Egg during Fertilization 2 The Role of Actin in Fertilization 4 Current Tools for Monitoring F-actin 5 Materials and Methods 7 Fish Maintenance and Breeding 7 Preparation of GFP-UtrCH Protein 7 SDS-PAGE and Coomassie Blue Staining 8 In vitro Fertilization (IVF) 9 Microinjecting Zebrafish Embryos and in Unfertilized Eggs 10 Rhodamine Phalloidin Staining 10 Generation of Tg(ziwi:GFP-UtrCH) 11 Time-lapse Recording during Fertilization 12 Results 13 Purification of GFP-UtrCH protein and application 13 The Diffuse Problem of GFP-UtrCH in Unfertilized Egg 14 Generation of Tg(ziwi:GFP-UtrCH) 14 Monitor the Changes of F-actin During Fertilization in Zebrafish 16 Discussion 18 References 21 Figures 25 | |
dc.language.iso | en | |
dc.title | 建立ziwi:GFP-UtrCH基因轉殖斑馬魚以監測受精卵肌動蛋白之變化 | zh_TW |
dc.title | Generation of ziwi:GFP-UtrCH Transgenic Zebrafish line for Monitoring Actin Dynamics in Fertilizing Eggs | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳俊宏(Jiun-Hong Chen),郭典翰(Dian-Han Kuo) | |
dc.subject.keyword | 斑馬魚,受精,肌動蛋白,基因轉殖, | zh_TW |
dc.subject.keyword | Zebrafish,Actin dynamics,Fertilization,Utrophin,Transgenic fish, | en |
dc.relation.page | 34 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2015-02-09 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生命科學系 | zh_TW |
顯示於系所單位: | 生命科學系 |
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