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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 | Hao-Hsiu Huang | en |
dc.contributor.author | 黃浩修 | zh_TW |
dc.date.accessioned | 2022-11-25T07:46:12Z | - |
dc.date.available | 2023-08-01 | |
dc.date.copyright | 2021-08-31 | |
dc.date.issued | 2021 | |
dc.date.submitted | 2021-08-09 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/82524 | - |
dc.description.abstract | CRISPR/Cas是細菌的後天免疫的防禦機制,用以防止其他細菌或嗜菌體的攻擊。其中第二型的CRISPR/Cas9更是被大家拿來廣泛的應用在基因編輯技術上,CRISPR/Cas9系統主要包含與目標基因匹配的guide RNA (gRNA) 以及能使雙股DNA斷裂的核酸內切酶Cas9。而Cas9 D10A nickase是一種只能將DNA剪斷一股的蛋白,同時有人提出使用double nicking的方法進行基因編輯時,可以降低脫靶效應 (off-target effects)。 當骨頭老化或是受損時,會進行骨骼重塑的修復機制,骨骼重塑包含成骨細胞的成骨作用及蝕骨細胞的骨吸收作用,兩種細胞息息相關互相調節及維持骨頭的動態平衡。當動態平衡被破壞就有可能造成一些骨頭相關的疾病,例如: 石骨症或骨質疏鬆等。因此了解成骨細胞與蝕骨細胞間的關係也就更為重要。現在已知進行骨頭重塑時成骨細胞會分泌化學訊號促使未成熟的蝕骨細胞分化形成成熟多核的蝕骨細胞進行骨吸收作用,去除壞死或老化的骨頭,成骨細胞再合成新的骨頭進行修復。 然而蝕骨細胞是否也會釋放訊號促使成骨細胞重建骨頭與其相關的機制尚未被釐清,因此本實驗目標是透過使用CRISPR/Cas9 D10A double nicking的基因編輯方法建立帶有蝕骨細胞專一表現之Acp5a (TRAP)的轉殖斑馬魚,以利後續藉由細胞摘除的方式探討蝕骨細胞的功能。實驗選擇用內含蝕骨細胞特有的Acp5a啟動子及Crystallin βb1 (Crbb1) 眼睛促進子,以及還原酶reductase和綠色螢光標記蛋白 (hrGFP),利用CRISPR/Cas9 D10A double nicking的方法建立Acp5a的轉殖斑馬魚。 在in vitro實驗的部分, 首先測試gRNA-16及gRNA-34與Cas9結合後,能否將線性化的Acp5a質體切出1400 bp左右的片段。同時也以先前建立帶有Six1啟動子附帶綠色螢光標記蛋白 (EGFP) 的質體作為對照組,測試gRNA-43及gRNA-77與Cas9結合後,能否將線性化的Six1質體切出700 bp左右的片段。由實驗結果確定gRNA-16及gRNA-34和gRNA-43及gRNA-77可以有效地將線性化的Acp5a質體及Six1質體切出1400 bp及700 bp的片段,由此推論in vivo實驗的可行性。 在in vivo實驗,將Cas9 D10A、gRNA、0.5 M KCl、phenol red以及水混和配製而成的sample,打入斑馬魚的受精卵,再進行觀察。在Six1,可在三天時看見綠色螢光訊號蛋白 (EGFP) 遍布在肌肉及耳石,並且發現有劑量效應。在Acp5a,可在3天時看見眼睛有螢光訊號蛋白 (hrGFP) 的表現,一週時可以在脊椎中間及尾巴看見蝕骨細胞的螢光訊號 (hrGFP),兩週可在尾柄看見蝕骨細胞的螢光訊號 (hrGFP) 。 另外由於質體還含有還原酶,因此未來可以利用還原酶將甲硝唑 (Metronidazole) 上的硝基還原成硝基咪唑 (Nitrosoimidazole) 誘導蝕骨細胞凋亡。在後續的實驗,可將轉殖斑馬魚進行泡藥摘除實驗,把魚浸泡在Metronidazole (MTZ) 中,觀察蝕骨細胞凋亡對骨細胞與骨頭型態的影響,並使用Alizarin red (染硬骨) 與alcian blue (染軟骨) 染色觀察硬骨與軟骨型態。也可以更進一步把各個蝕骨細胞表現之時間點,進行泡藥摘除細胞實驗,且染色觀察斑馬魚各階段蝕骨細胞的摘除對骨頭發育的影響。 | zh_TW |
dc.description.provenance | Made available in DSpace on 2022-11-25T07:46:12Z (GMT). No. of bitstreams: 1 U0001-0608202112215700.pdf: 4670288 bytes, checksum: 167c8b926b1937e4032a65a9d6fa4b8d (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 英文摘要 v 壹、 前言 1 貳、 實驗材料 32 參、 實驗方法 38 肆、 實驗結果 51 伍、 討論 57 陸、 未來方向 60 柒、 圖表 61 捌、 參考文獻 78 | |
dc.language.iso | zh-TW | |
dc.title | 建立CRISPR/Cas9基因轉殖系統及Acp5a轉殖斑馬魚 | zh_TW |
dc.title | Establishment of CRISPR/Cas9 gene targeting system and its application to Tg(acp5a:reductase-IRES-hrGFP) zebrafish | 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 | 蝕骨細胞,TRAP(Acp5a),CRISPR/Cas9,Cas9 D10A,斑馬魚,double nicking, | zh_TW |
dc.subject.keyword | osteoclast,TRAP(Acp5a),CRISPR/Cas9,Cas9 D10A,paired nickase,zebrafish, | en |
dc.relation.page | 85 | |
dc.identifier.doi | 10.6342/NTU202102142 | |
dc.rights.note | 同意授權(限校園內公開) | |
dc.date.accepted | 2021-08-09 | |
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 | 2023-08-01 | - |
顯示於系所單位: | 口腔生物科學研究所 |
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