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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 陳佑宗(You-Tzung Chen) | |
| dc.contributor.author | Kuo-Hung Lee | en |
| dc.contributor.author | 李國宏 | zh_TW |
| dc.date.accessioned | 2021-06-17T06:00:38Z | - |
| dc.date.available | 2019-03-05 | |
| dc.date.copyright | 2019-03-05 | |
| dc.date.issued | 2019 | |
| dc.date.submitted | 2019-02-12 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/71433 | - |
| dc.description.abstract | WD Repeat and HMG-Box DNA Binding Protein 1(WDHD1)在真核生物中為一個演化上保守的蛋白，先前文獻研究指出，WDHD1在DNA複製起始(initiation)及延長(elongation)機制上扮演了重要的角色，WDHD1就如同一個中心樞紐的鷹架，幫助其他與DNA複製相關複合體的形成(如:pre-replication complex以及replication protein complex)，並增加DNA複製的效率，藉此來維持整個基因體的完整性。演化上，脊椎動物的WDHD1相較於非脊椎動物，除了都有WD40及SepB功能區域(functional domain)之外，還多了HMG-box 區域。當人類細胞WDHD1缺失時會造成細胞無法成功地完成細胞週期以及造成染色體分離異常。在本篇論文中，我們以CRISPR/Cas9基因修改的技術產生了Wdhd1缺失的小鼠，來研究其於哺乳類動物活體內之功能，同時也將針對HMG-box區域的功能進行探討。目前已產生出攜帶於Wdhd1第一個外顯子有移碼突變(c.13delinsTGAGA)等位基因的小鼠，觀察到帶有該突變之同型合子會死於胚胎發育早期，並且於E7.5到E8.5之間有明顯的生長異常的外觀。Wdhd1 c.13delinsTGAGA同型合子胚胎在著床前階段(pre-implantation stage)顯示出明顯正常的生長，但在E7.5階段則觀察到生長異常的胚胎。進一步的研究則發現，在Wdhd1 c.13delinsTGAGA同型合子胚胎的E7.5階段，細胞增殖率降低並且有細胞凋亡的情形。總而言之，這些發現揭示了Wdhd1對小鼠胚胎發育至關重要，一旦Wdhd1缺失會導致 E7.5-E10.5胚胎生長異常，最終導致了胚胎致死。 | zh_TW |
| dc.description.abstract | WD Repeat and HMG-Box DNA Binding Protein 1(WDHD1) is an evolutionary conserved protein in eukaryotes. Previous research indicates that WDHD1 plays a crucial role in initiation and elongation of DNA replication. WDHD1 acts as a hub to help the formation of DNA replication-related complexes (e.g., pre-replication complex and replication protein complex) and it also ensures efficient DNA replication. WDHD1 is required for gnome integrity maintenance. Phylogenetic analysis revealed that, compared to non-vertebrate eukaryotes, vertebrate WDHD1 not only possesses a WD40 domain and a SepB domain but also has an additional HMG-box domain at its C-terminus. WDHD1 deficiency in human cell causes cell cycle delays and abnormal chromosome segregations. In order to study its biological function in mammal in vivo, we use the CRISPR/Cas9 gene editing tool to generate Wdhd1 deficient mice. In addition, we are going to study the HMG-box domain of Wdhd1. A Wdhd1 exon1 frameshift mutation (c.13delinsTGAGA) in the mouse was generated and found that this mutant allele can cause embryonic lethality when it is bred to homozygosity and discovered abnormal phenotype of embryos at the stage of E7.5 and E8.5. Wdhd1 c.13delinsTGAGA homozygous mutant embryos showed apparently normal growth during the pre-implantation stage, but retarded growth at E7.5. Further investigation showed that the cell proliferation ratio decreased and also showed apoptosis signal at E7.5 homozygous mutant embryos. Taken together, these findings revealed that Wdhd1 is essential for mouse embryogenesis. Once Wdhd1 is defective, it causes abnormal growth of the embryo at E7.5-E10.5 and eventually leads to embryonic lethality. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T06:00:38Z (GMT). No. of bitstreams: 1 ntu-108-R05455004-1.pdf: 3227190 bytes, checksum: beb9fcd559ef4d74c38a43130e7b0466 (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 口試委員審定書…………………………………………………………………………i
致謝………………………………………………………………………………….......ii 中文摘要………………………………………………………………………………iii Abstract………………………………………………………………………………….iv Contents………………………………………………………………………………….v List of figures…………………………………………………………………………vii List of tables……………………………………………………………………………ix List of supplementary tables……………………………………………………………x 1. Introduction……………………………………………………………………….…1 1.1 The function of WDHD1 in DNA replication ………...……….……..…….…1 1.2 Evolution divergence of WDHD1 in eukaryotes………………..…….………3 1.3 Genetically engineered mouse model for research………………...………..…4 1.4 The tool for gene editing: CRISPR/Cas9 system…………………..…….……5 2. Materials and methods………………………………………………………………8 2.1 SgRNA designs and spCas9 plasmid ……………………………………….…8 2.2 Cell culturing and transfection………….…………………………………..…8 2.3 The analysis of indel efficiency ……………………………………………….9 2.4 Western blotting……………………………………….…………………….10 2.5 Dissection and in vitro culturing of pre-implantation embryos………………11 2.6 Genotyping………….…….…………………………………………………12 2.7 RNA isolation….……………….……………………………………………12 2.8 Reverse transcription and Quantitative real time PCR…………………….…13 2.9 EdU cell proliferation assay.………………………….……………………...14 2.10 Paraffin section of E7.5 mouse embryos…………………..…………………15 2.11 BrdU proliferation assay……………………………………………………..15 2.12 TUNEL assay……….…………………………………………………...…..16 2.13 Generation of Wdhd1 mutant mice………………………………………......17 2.14 Statistical analysis…………………………………………………………...17 3. Results……………………………………………………………………………..19 3.1 SgRNAs targeting Wdhd1 and their functional validation in vitro……..…….19 3.2 The generation of Wdhd1 mutant mice………………………………………20 3.3 Wdhd1 c.13delinsTGAGA homozygotes result in embryonic lethality….......21 3.4 Wdhd1 expression level at different embryonic stages….................................22 3.5 Wdhd1 mutation affected cell proliferation rate and caused cell apoptosis in E7.5 mouse embryos…………………………………………………………23 4. Discussion………………………………………………………………………….25 5. References…………………………………………………………………………29 | |
| dc.language.iso | zh-TW | |
| dc.title | Wdhd1於小鼠早期胚胎發生之功能性研究 | zh_TW |
| dc.title | The functional study of Wdhd1 in mouse early embryogenesis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 107-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張以承(Yi-Cheng Chang),游益興(I-Shing Yu),陳俊銘(Chun-Ming Chen) | |
| dc.subject.keyword | DNA複製,細胞增生,Wdhd1,CRISPR/Cas9,小鼠, | zh_TW |
| dc.subject.keyword | DNA replication,cell proliferation,Wdhd1,CRISPR/Cas9,mouse, | en |
| dc.relation.page | 54 | |
| dc.identifier.doi | 10.6342/NTU201900448 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2019-02-12 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 基因體暨蛋白體醫學研究所 | zh_TW |
| Appears in Collections: | 基因體暨蛋白體醫學研究所 | |
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| ntu-108-1.pdf Restricted Access | 3.15 MB | Adobe PDF |
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