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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 丁詩同(Shih-Torng Ding) | |
dc.contributor.author | Yu-Hui Pan | en |
dc.contributor.author | 潘玉惠 | zh_TW |
dc.date.accessioned | 2021-06-16T10:22:08Z | - |
dc.date.available | 2018-08-28 | |
dc.date.copyright | 2013-08-28 | |
dc.date.issued | 2013 | |
dc.date.submitted | 2013-08-16 | |
dc.identifier.citation | 張慧卉。2012。不飽和脂肪酸降低鴨脂肪酸延長酶與去飽和酶之基因表現。國立台灣大學生物資源暨農學院動物科學技術學系碩士論文。pp. 13-19。
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Yolk sac endoderm is the major source of serum proteins and lipids and is involved in the regulation of vascular integrity in early chick development. Development Dynamics 240: 2002-2010. Noble, R., and M. Cocchi. 1990. Lipid metabolism and the neonatal chicken. Progress in lipid research 29: 107-140. Ono, T., R. Yokoi, and H. Aoyama. 1996. Transfer of male or female primordial germ cells of quail into chick embryonic gonads. Experimental Animals 45: 347-352. Park, T. S., M. A. Kim, J. M. Lim, and J. Y. Han. 2008. Production of quail (Coturnix japonica) germline chimeras derived from in vitro‐cultured gonadal primordial germ cells. Mol Reprod Dev 75: 274-281. Powell, K. A., E. A. Deans, and B. K. Speake. 2004. Fatty acid esterification in the yolk sac membrane of the avian embryo. Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology 174: 163-168. Poynter, G., D. Huss, and R. Lansford. 2009. 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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/60575 | - |
dc.description.abstract | 鳥類胚胎發育時從卵白及卵黃中獲得營養,其中卵黃中膽固醇係以游離膽固醇形式儲存,而在轉移至卵黃囊膜時被酯化為膽固醇酯。在胚胎發育後期,卵黃囊膜中固醇轉醯酶 (soat, Sterol-O-acyltransferase)活性提高,顯示固醇轉醯酶可能在營養分利用上扮演重要的角色,因此本論文的目的在建立轉基因鵪鶉模式,以探討卵黃囊膜中固醇轉醯酶在鳥類胚胎發育時的功能。
固醇轉醯酶有兩個異構型-固醇轉醯酶1與固醇轉醯酶2,兩種異構型均有酯化膽固醇的能力。為了確認何者為卵黃囊膜中主要的固醇轉醯酶,我們分析了不同階段的雞與日本鵪鶉胚胎,結果顯示,固醇轉醯酶1的mRNA表現量在胚胎發育後期有急遽增加的現象,而固醇醯酶2則沒有顯著變化並且於孵化過程中維持低量的表現。因此,我們推測固醇轉醯酶1為胚胎發育時主要的固醇轉醯酶,並以雞的基因組DNA為模板,選殖固醇轉醯酶1的調控區域並構築於綠螢光表現慢病毒載體中,將構築好的轉基因載體轉染雞與鵪鶉原代肝臟細胞,結果二者均有綠螢光的表現,證實選殖出的固醇轉醯酶1調控區域具有在肝臟細胞中表現的能力。 另一方面,我們首先使用pLenti-CAG-eGFP質體,藉293T細胞產製高力價病毒,利用注射慢病毒於stage X的日本鵪鶉胚胎的胚盤下腔方式建立轉基因鵪鶉模式。一共注射628個胚胎,經16-18天後孵化。以螢光顯微鏡檢視孵化蛋殼上絨毛尿囊膜的綠螢光表現確認轉基因結果,結果獲得6隻小鵪鶉,其中四隻為成功嵌合體轉基因鵪鶉。將其中一隻各組織冷凍切片,發現許多組織都有轉基因綠螢光蛋白的表現。四隻轉基因鵪鶉中僅一隻公鵪鶉存活到性成熟,將其與三隻野生型鵪鶉進行雜交。收集50個種蛋進行孵化。以專一性引子檢測其絨毛尿囊膜中eGFP的有無,結果顯示,我們成功地產製許多具有性腺傳承及外源綠螢光蛋白質表現的親代轉基因鵪鶉。 綜合上述,我們成功地選殖到了固醇轉醯酶1的調控區域並且建立了轉基因鵪鶉模式,以此模式為基礎,未來可應用於基因功能研究或作為生物反應器產製不同的生醫材料。 | zh_TW |
dc.description.abstract | During incubation, the developing embryo obtains nutrients from albumen and yolk. Initially, cholesterol is stored as free cholesterol in the yolk, and can be esterified into cholesteryl esters after transferring into yolk sac membrane (YSM). During the second half of embryonic development, sterol O-acyltransferase (soat, also referred as acat) activity in the YSM increases dramatically, suggesting that soat may play an important role for cholesterol and fatty acid transportation. Since there are much unknown about cholesterol transport between yolk, YSM and embryo liver, to study the function of soat in YSM during avian embryo development, we generated a soat overexpression avian model.
Two soat isoforms, soat1 and soat2 were identified. Both of the two enzymes have the ability to esterify cholesterol. To identify the soat subtype is the major enzyme in the YSM, Japanese quail embryos of different developing stages were sacrificed to examine different gene expression. Results showed that soat1 is the major form in YSM and the mRNA expression increased dramatically during the second half of embryo development. Then, we cloned soat1 promoter from the chicken genomic DNA and then constructed into lentiviral vector with a reporter gene, enhanced green fluorescence protein (eGFP). The eGFP expression was detectable in both chicken and quail primary hepatocytes after lentiviral infection, confirming that the downstream target, eGFP, is driven by the promoter in the livers in these species. We then created another transgenic model, Japanese quail by using the lentiviral vector containing eGFP as a reporter driven by chicken β-actin promoter. We injected concentrated lentivirus (titer >1×108 transforming units/mL) into the subgerminal cavity of freshly laid eggs at stage X to infect primordial germ cells originated in the area pellucida. After 16-18 days of incubation, we got 6 chicks from 628 quail eggs with 4 of them containing a mosaic eGFP expression. Only one male mosaic founder (G0) survived to maturity. eGFP expression was found in the heart, lung, liver, kidney, colon, muscle, gizzard, hair, and skin tissues in one dead G0 quail. The matured male G0 was mated with three female nontransgenic wild-type female and totally 50 fertilized eggs were collected. Phenotypic screening showed the expression of eGFP in chorioallantoic membrane (CAM) in the G1 quails. To confirm the integration of the transgene in G1 quails, genomic DNA was extracted from the CAM and the primer set for the 409-bp fragment was used to detect the transgene using PCR. Results suggested that the mosaic founder bears the ability of germ-line transmission of the transgene. In conclusion, we successfully cloned soat1 promoter and established a transgenic quail model. This model can be used to study gene function or to generate biomedical products for various purposes. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T10:22:08Z (GMT). No. of bitstreams: 1 ntu-102-R00626002-1.pdf: 3049887 bytes, checksum: 48b5f0801f8d8aa4c31f1b48ba69052e (MD5) Previous issue date: 2013 | en |
dc.description.tableofcontents | 目錄
中文摘要 II Abstract IV 圖目錄 VI 表目錄 VII 第壹章、文獻回顧 1 壹、 禽類胚胎營養傳遞 1 一、 卵黃囊膜 (yolk sac membrane, YSM) 1 二、 YSM中膽固醇的再酯化 3 貳、 Sterol O-acyltransferase 1 的功能與結構 4 參、 基因轉殖禽類模式 5 一、 始基生殖細胞 (primordial germ cells, PGC) 法 5 二、 精子載體(sperm-mediated gene transfer, SMGT) 法 6 三、 慢病毒載體(Lentivirus) 法 7 第貳章 材料與方法 9 壹、 雞胚soat1的表現 10 一、 受精蛋培養與採集 10 二、 soat1與soat2基因表現 10 貳、 soat1 promoter 選殖 11 一、 雞基因組DNA的萃取 11 二、 預測soat1啟動子區域和聚合酶鏈鎖反應之引子設計 12 三、 PCR產物接合與轉形 13 四、 質體截切與慢病毒載體構築 13 參、 Soat1 基因選殖 13 肆、 pLenti-soat1p-eGFP功能評估 15 一、 雞肝臟細胞原代培養 (chicken liver primary culture) 15 二、 鵪鶉肝臟細胞原代培養 15 三、 pLenti-soat1p-eGFP轉染(transfection)肝臟細胞 16 伍、 轉基因日本鵪鶉模式建立 16 一、 日本鵪鶉受精蛋之收集 16 二、 高力價慢病毒 ( High titer Lentivirus)備製 16 三、 慢病毒感染鵪鶉胚胎 18 四、 轉基因檢測 19 陸、 統計方式 20 第參章 結果與討論 21 壹、 雞胚胎與鵪鶉胚胎soat1的表現 21 貳、 soat1 promoter 選殖 24 一、 soat1啟動子預測區域之選殖 24 二、 質體截切與慢病毒載體構築 25 三、 soat1 CDS 選殖 26 參、 pLenti-soat1p-eGFP功能評估 28 肆、 轉基因日本鵪鶉模式建立 30 第肆章 結論 36 第伍章 參考文獻 37 | |
dc.language.iso | zh-TW | |
dc.title | 建立轉基因鵪鶉模式探討固醇轉醯酶在胚胎的功能 | zh_TW |
dc.title | Establishment of a transgenic quail model for studying the potential function of soat1 | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 林恩仲(En-Chung Lin) | |
dc.contributor.oralexamcommittee | 吳信志(Shinn-Chih Wu),張文興(Wen-Hsing Chang),陳洵一(Shuen-Ei Chen) | |
dc.subject.keyword | Sterol-O-acyltransferase,轉基因鵪鶉,慢病毒,卵黃囊膜,膽固醇酯,禽類胚胎, | zh_TW |
dc.subject.keyword | Sterol O-acyltransferase 1,transgenic quails,lentivirus,yolk sac membrane,cholesteryl ester,bird embryos, | en |
dc.relation.page | 39 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2013-08-16 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 動物科學技術學研究所 | zh_TW |
顯示於系所單位: | 動物科學技術學系 |
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