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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張百恩(Bei-En Chang) | |
dc.contributor.author | Shiang-Hsuan Lee | en |
dc.contributor.author | 李享軒 | zh_TW |
dc.date.accessioned | 2022-11-25T08:01:24Z | - |
dc.date.copyright | 2021-08-31 | |
dc.date.issued | 2021 | |
dc.date.submitted | 2021-08-10 | |
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Using the stomach-distance ratio as a possible diagnostic tool for prediction of right atrial isomerism. Ann Acad Med Singap 37, 906-912. 50.Yelon, D., and Stainier, D.Y. (1999). Patterning during organogenesis: genetic analysis of cardiac chamber formation. Semin Cell Dev Biol 10, 93-98. 51.Yosef, I., Goren, M.G., and Qimron, U. (2012). Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res 40, 5569-5576. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/82870 | - |
dc.description.abstract | "兩側右心房症(Right Atrial isomerism, RAI)為異位綜合症(Heterotaxy syndrome)的一種,是在台灣有特別高的發生機率複雜型先天心臟疾病。在胚胎發育時期,左右不對稱的過程發生了問題,使得身體的兩側皆發育成類似於右側的器官。病患有兩個右心房,沒有左心房,另外常合併有單一心室,右心室雙出口,肺靜脈回流異常,心內膜墊缺損和心律不整等問題。 儘管在台灣有較高的發生機率,這一類病患背後的遺傳訊息仍然很少被研究,我們和台大醫院小兒科林銘泰醫師合作,林醫師團隊將臨床病患檢體運用NGS (Next generation sequencing)的技術分析,已從46名RAI病患中發現了新的Nodal基因突變(R307Q)。 Nodal是轉化生長因子(TGF-β) subfamily的成員,在胚胎原腸化(gastrulation)過程中為調節中胚層和內胚層特化的誘導信號,在左右不對稱發育中扮演重要的角色。另外也參與了神經外胚層前後軸線的分化。不同於哺乳動物,斑馬魚有兩個Nodal 基因(ndr1、ndr2),轉譯成兩個蛋白質: Nodal-related 1 (NDR1)和Nodal-related 2 (NDR2),產生的Nodal信號會使囊胚期動物極邊緣細胞發育成中胚層和內胚層。這兩個蛋白質發生突變可能會導致Nodal信號途徑(Nodal signaling pathway)發生異常,從而影響斑馬魚胚胎發育左右不對稱決定過程發生異常,造成心臟畸形或缺陷等症狀。 為了研究Nodal基因突變(R307Q)和臨床上表現型有何關聯,我們使用CRISPR-Cas9的系統將Nodal(R307Q) knock in在斑馬魚模式動物上。不同於一般CRISPR系統,我們選擇使用Cas9 Nickase而非Cas9 Nuclease,利用Cas9 Nickase來進行基因編輯,需要兩個gRNA而不是一個。這兩個gRNA將被設計在相反的DNA strand上且緊密接近,序列相距不能超過20bp,以確保一旦兩條strand被Cas9 Nickase切開時,DNA double strand才斷開。這種配對的Cas9 Nickase比起Nuclease減少了脫靶效應(off-targeting),因為兩個gRNA需要一起作用才能將DNA double strand斷開,後續同源介導的雙鏈DNA修復(homology directed repair)路徑將被激活以完成基因編輯過程。 在備製CRSPR系統中做為Donor DNA的質體中,除了要knock in進入的Nodal-related 1 (R307Q)片段外,在Exon 3還附帶綠色的螢光標記蛋白(Hu-Cr-IRES-GFP)並且在兩側加入含有gRNA辨識切位的oilgomers來做斑馬魚轉殖實驗。 在我實驗中可以發現NDR1有表現的位置大致分佈在脊索、神經管和尾部等地方,將斑馬魚魚卵運用microinjection技術注入CRSIPR-Cas9系統(gRNA、Donor DNA and Cas9 Nickase)來基因轉殖斑馬魚,約三天後可以觀察到魚眼睛水晶體內有綠色螢光表現,頭部腦後神經管以及尾部都有螢光訊號發現。除此之外F0有觀察斑馬魚會有畸形的情形發生,包括心臟也有不正常looping的表現。 未來實驗F1可以比較有成功knock in斑馬魚和wild type斑馬魚心臟形態差異,計算心跳數作為是否有心律不整的比較等,並試著成功將斑馬魚養大配種成穩定的品系(stable line),當作人類兩側右心房症(Right Atrial isomerism, RAI)疾病的動物模型(animal model)。" | zh_TW |
dc.description.provenance | Made available in DSpace on 2022-11-25T08:01:24Z (GMT). No. of bitstreams: 1 U0001-0908202114323000.pdf: 6226574 bytes, checksum: 74abe769bee4dc8052d2417e06a589e4 (MD5) Previous issue date: 2021 | en |
dc.description.tableofcontents | "口試委員會審定書...i 致謝...ii 中文摘要...iii Abstract...v 一、異位綜合症(Heterotaxy syndrome)...8 二、兩側右心房症(Right Atrial isomerism, RAI)...8 三、兩側右心房症的病因學(Etiology of RAI)...10 四、臨床檢體研究(clinical specimen research)...12 五、斑馬魚ndr1 mRNA的分布...13 六、斑馬魚疾病動物模型和其心臟發育...18 七、CRISPR...21 八、CRISPR Cas9選擇(Nickase and Nuclease)...27 九、研究動機...29 貳、實驗材料...30 參、實驗方法...37 肆、實驗結果...68 伍、討論...72 陸、圖表...75 柒、參考文獻...113" | |
dc.language.iso | zh-TW | |
dc.title | 利用CRISPR基因編輯方法在斑馬魚建立人類疾病模式:以Nodal mutation為範例 | zh_TW |
dc.title | Modeling human disease in zebrafish by CRISPR gene editing:Nodal mutation as a paradigm | 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 | Right Atrial isomerism (RAI),Nodal-related 1 (NDR1),CRISPR,Cas9 Nickase,斑馬魚, | zh_TW |
dc.subject.keyword | Right Atrial isomerism (RAI),Nodal-related 1 (NDR1),CRISPR,Cas9 Nickase,zebrafish, | en |
dc.relation.page | 119 | |
dc.identifier.doi | 10.6342/NTU202102209 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2021-08-10 | |
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 | 2023-08-09 | - |
顯示於系所單位: | 口腔生物科學研究所 |
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