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Title: | 建立轉基因鵪鶉模式探討固醇轉醯酶在胚胎的功能 Establishment of a transgenic quail model for studying the potential function of soat1 |
Authors: | Yu-Hui Pan 潘玉惠 |
Advisor: | 丁詩同(Shih-Torng Ding) |
Co-Advisor: | 林恩仲(En-Chung Lin) |
Keyword: | Sterol-O-acyltransferase,轉基因鵪鶉,慢病毒,卵黃囊膜,膽固醇酯,禽類胚胎, Sterol O-acyltransferase 1,transgenic quails,lentivirus,yolk sac membrane,cholesteryl ester,bird embryos, |
Publication Year : | 2013 |
Degree: | 碩士 |
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。 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/60575 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 動物科學技術學系 |
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