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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張百恩(BEI-EN CHANG) | |
| dc.contributor.author | Hong-Xuan Lu | en |
| dc.contributor.author | 盧弘軒 | zh_TW |
| dc.date.accessioned | 2022-11-25T07:45:48Z | - |
| dc.date.available | 2023-08-31 | |
| dc.date.copyright | 2021-09-16 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-08-23 | |
| dc.identifier.citation | Albadri, S., Del Bene, F., Revenu, C. (2017). Genome editing using CRISPR/Cas9-based knock-in approaches in zebrafish. Methods, 121-122, 77-85. https://doi.org/10.1016/j.ymeth.2017.03.005 Arnaout, R., Ferrer, T., Huisken, J., Spitzer, K., Stainier, D. Y., Tristani-Firouzi, M., Chi, N. C. (2007). Zebrafish model for human long QT syndrome. Proc Natl Acad Sci U S A, 104(27), 11316-11321. https://doi.org/10.1073/pnas.0702724104 Asnani, A., Peterson, R. T. (2014). The zebrafish as a tool to identify novel therapies for human cardiovascular disease. Dis Model Mech, 7(7), 763-767. https://doi.org/10.1242/dmm.016170 Augustine, S., Gagnaire, B., Floriani, M., Adam-Guillermin, C., Kooijman, S. A. (2011). Developmental energetics of zebrafish, Danio rerio. Comp Biochem Physiol A Mol Integr Physiol, 159(3), 275-283. https://doi.org/10.1016/j.cbpa.2011.03.016 Bakkers, J. (2011). Zebrafish as a model to study cardiac development and human cardiac disease. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/82498 | - |
| dc.description.abstract | Right atrial isomerism (RAI) 又稱兩側右心房症，屬於一種先天性心臟病，新生兒發生率大概約0.02~0.05%，如果不治療，致死率極高。一般的正常人心臟會有4個腔室，包含左右的心房和心室，但RAI的病患只有一個右心房和一個右心室，這會導致充氧血和缺氧血無法阻隔且混合，血氧濃度偏低，出現發紺的缺氧現象。發生的主因是因為在胚胎形成時，控制左右器官分化的基因突變，導致身體無法正常分化，變成兩邊都是右邊的器官，且常合併無脾症。 我們在RAI患者抽血，然後把血液樣本拿去做NGS次世代定序，結果發現Nodal基因有突變發生，307位置的精胺酸 (Arginine) 發生missense變成麩醯胺酸 (Glutamine) (R307Q)。Nodal基因是TGFβ家族的一員，位於10號染色體上，與早期胚胎的發育相關，在人類中，Nodal的突變可能導致異位綜合症 (Heterotaxy)。在斑馬魚中可分為ndr1 (squint mutant) 和ndr2 (cyclops mutant)，此實驗是將一段突變的序列－ndr2 (R307Q)，以及Crystallin眼睛促進子，附帶綠色的螢光標記蛋白的質體，利用CRISPR/Cas9的方式進行斑馬魚轉殖實驗，爾後觀察斑馬魚的心臟發育。 CRISPR/Cas9是一種細菌遭噬菌體入侵的防禦機制，後來被應用於基因編輯技術上，在此CRISPR/Cas9系統，我們使用了Cas9 D10A Nickase，並提供兩條guide RNA (gRNA)，透過double nicking的方法進行基因編輯，藉此降低脫靶效應(off-target effects)。 首先在in vitro實驗，測試gRNA-58號和gRNA-71號以及Cas9 Nuclease，是否能將pGEMT-R307R-1st (ScaI線性化) 和pGEMT-R307Q-1st (ScaI線性化) 和BS-L-oligo-ndr2-exon2-intro2-exon3-R307R-Hu-Cr-IRES-GFP-3'-UTR-R-oligo-1st質體 (XhoI和EcoRI線性化) 切斷。結果顯示，gRNA-58號都會切所有測試的。但是，兩個核苷酸突變 (R307Q) 之後，gRNA-71號依然會辨識R307Q，且切斷DNA，表示有脫靶效應(off-target effects)。 由於上述的效應，我們在兩個gRNA對應的位置進行多點核甘酸序列的突變 (wobble mutation)，避免脫靶效應，得到pGEMT-R307R-2nd和pGEMT-R307Q-2nd，這兩個質體，測試結果都不會被gRNA-58號和gRNA-71號所辨識，因此可進行後續in vivo的顯微注射實驗。 在in vivo的實驗，以顯微注射的方式將兩個gRNA、Cas9 D10A Nickase、donor DNA打進一個細胞期的斑馬魚受精卵，爾後進行觀察。在F0第四天的胚胎中，觀察到綠色的螢光蛋白在神經管、neuromast、floor plate以及尾巴區域零星表現。經過篩選，F1的穩定品系中，在第五天時，觀察到綠色的螢光蛋白在神經細胞、floor plate、neuromast零星表現。F1綠色螢光表現的位置，是否對應ndr2基因表現的位置，有待釐清。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-25T07:45:48Z (GMT). No. of bitstreams: 1 U0001-1808202122084500.pdf: 25201122 bytes, checksum: 9cdc1a117df94f1c1cfabd5bf6998bcd (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | 口試委員審定書 i 致謝 ii 中文摘要 iii ABSTRACT v 壹、 前言 1 一. 斑馬魚模式動物以及其胚胎發育過程 1 二. 兩側右心房症的臨床表現 2 三. 兩側右心房症的病因 3 四. 臨床檢體研究 6 五. 斑馬魚中NODAL的分佈 8 六. 在斑馬魚動物模型中研究人類心臟疾病 12 七. 斑馬魚心臟的發育與形態 13 八. 斑馬魚在胚胎以及成魚階段時的心率 17 九. CRISPR的由來 18 十. CRISPR/CAS9的作用機制與應用 21 十一. CRISPR/CAS9、ZEN、TALEN三者之間的比較 23 十二. CAS9 D10A NICKASE可降低脫靶效應 24 十三. 使用CRISPR/CAS9系統建立穩定的基因轉殖魚 26 十四. 研究動機 28 貳、 實驗材料 30 參、 實驗方法 39 肆、 結果 74 伍、 討論 81 陸、 未來方向 84 柒、 圖表 85 捌、 參考文獻 114 | |
| dc.language.iso | zh-TW | |
| dc.title | 在斑馬魚以CRISPR/Cas9基因編輯方法建立ndr2突變之人類遺傳疾病模式 | zh_TW |
| dc.title | CRISPR/Cas9 gene editing of zebrafish ndr2 mutation for modeling human disease | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 侯欣翰(Hsin-Tsai Liu),林銘泰(Chih-Yang Tseng) | |
| dc.subject.keyword | Nodal,cyclops,兩側右心房症,CRISPR/Cas9,Cas9 D10A Nickase,double nicking,斑馬魚, | zh_TW |
| dc.subject.keyword | Nodal,cyclops,right atrial isomerism,CRISPR/Cas9,Cas9 D10A Nickase,double nicking,zebrafish, | en |
| dc.relation.page | 120 | |
| dc.identifier.doi | 10.6342/NTU202102494 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2021-08-23 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 口腔生物科學研究所 | zh_TW |
| dc.date.embargo-lift | 2023-08-31 | - |
| 顯示於系所單位： | 口腔生物科學研究所 | |
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