https://scholars.lib.ntu.edu.tw/handle/123456789/199585
標題: | 行政院國家科學委員會專題研究計畫成果報告:基因槍在眼表層細胞中之研究 | 作者: | 胡芳蓉 | 關鍵字: | 角蛋白;基因槍;專題計畫;報告格式;國科會;Research Project;Report Style;National Science Council | 公開日期: | 1999 | 出版社: | 臺北市:國立臺灣大學醫學院眼科 | 摘要: | keratin 3 和12(k3/12)是只表現在眼球 表面的一對特異纖維蛋白質(intermediate filament),而調節k3 和k12 之基因表現, 例如cis-DNA regulatory element,目前尚在 研究當中。目前的研究當中尚未有定論在 k12 cis-DNA regulatory element 上游需多 大的DNA 序列才能有效的表現出其調節 能力。而且k12 的promoter 無法有效轉殖 在primary conjunctival epithelial cell 中,雖 然在subculture 之細胞或是SV40 T antigen transformed 之conjunctival epithelial cell 中卻可以有效的轉殖,但是在這些細胞中 無法有效的表現k12 蛋白質。但是以k12 promoter-b-galactosidase DNA construct 轉 殖在這些細胞中發現k12 之5’-端上游0.2 kb 的cis-DNA 可以有效的表現出其 promoter 之活性。但是在transgenic mouse 的實驗卻無法有效表現其活性。 為了解決這一個問題,我們設計以in vivo particle-mediated gene transfer(‘’HeliosTN Gene Gun System’’)技 術來測試k12 promoter-reporter construct 的 活性。但是為了進一步鑑定更長的promoter DNA 序列以便進行transgenic mouse 或in vivo transfected rabbits 的實驗。而經由此模 式我們可以暸解k12 基因的調控,而能將 此實驗結果運用於k12-reporter construct 之 transgenic mouse 或transfected rabbits 之實 驗當中。進而製造眼角膜的k12 promoter 和一些growth factors 或cytokine 的chimera 應用於眼表層疾病的研究和治療。 本計畫在美國辛辛那提大學醫院眼科高惠 陽博士回國擔任本計畫之共同主持人指導 之下,在k12 Gene Gun in vivo transfection assay 的實驗上己有初步的結果,我們發現 在位於k12 promoter 上游的4 對Pax-6 pair binding elements 對調控k12 gene 在角膜上 的表現有相當大的影響,當我們delete 掉 k12 promoter 上的Pax-6 elements 時k12 在 角膜上的表現就變弱了,同時在結膜和皮 膚上的基因轉殖則沒有差別。所以我們認 為Pax elements 對k12 基因在角膜上的表 現相當重要。k12 一般只能表現在角膜表皮 細胞中,而不能表現在結膜細胞和培養之 角膜細胞中,所以它是具有角膜特異性的 一種基因。而是否Pax-6 在角膜細胞中伴 演著控制其器官特異性分化之作用或是有 其它transcriptional factor 影響其表現是接 下來我們所想暸解的事情。 The regulatory cis-DNA elements of the cornea-specific keratin 12 gene has been studied for a long time. Conventional ways of identifying this cell-specific promoter using cultured rabbit corneal epithelial cells derived from cornea explants is studied by many other authors and had following conclusions. First, for unknown reasons , these primary corneal epithelial cells could not be effectively transfected using liposomes. Second, the subcultured epithelial cells from cornea explants could be transfected but they dedifferentiated and ceased to express keratin 12. Third, SV40 large T-antigen transformed rabbit corneal epithelial cell line is also used to test K12 promoter-b-gal DNA constructs, because they could be efficiently transfected by liposomes or electroporation. This established 2 corneal epithelial cell line maintains many epithelial cell characteristics, but they no longer express keratin 12. However, this cell line is still of value to examlne K12 promoter- b-gal DNA constructs for their functionality, because it is easy to maintain and still possesses many epithelial cell characteristics, e.g. stratification, expression of keratins, etc. From the studies of Dr. Kao, a 0.2 kb element in the 5'-flanking region of the keratin 12 gene is identified that could direct the expression of b-galactosidase by using this cell line. As the 5'-end flanking sequences increased in the promoter-reporter gene constructs, the levels of b-galactosidase expression decreased in the transfected rabbit corneal epithelial cells. These observations imply that 5’ end sequence of the Krt1.12 gene may contain sufficient and essential regulatory cis-DNA elements for corneal epithelial cell-specific expression of the reporter gene(s), e.g. CAT (chloroamphenicol acetyltransferase) and b- galactosidase. Thus, a selective group of the reporter DNA constructs is tested with the transgenic mouse technique. Unfortunately, none of the transgenic mouse lines expressed reasonable levels of the CAT activity in cornea or other tissues. In order to circumvent these problems, we have tried the newly developed in vivo particle-mediated gene transfer techniques (Helios™ Gene Gun System") for a few trials to examlne the promoter activities of reporter gene constructs. The results of these "Gene Gun" experiments indicate that our reporter gene constructs (1.0 KZ, 2.5 KZ) contain regulatory cis-DNA elements for cornea epithelial cell expression of b-galactosidase. However, more experiments are needed to identify and characterize the DNA elements which are essential and sufficient for cornea epithelial cell-specific expression of reporter gene(s). Thus, the purpose of this study is to apply the "Gene Gun" system in the study of ocular surface epithelium. Then we can identify the exact structure of cis-regulatory DNA element of Krt1.12 gene. The identification of such DNA elements is a prerequisite for creating transgenic mouse models which may over-express cytokines, or carry domlnant negative mutations of receptors for future studies of corneal physiology, and development of a gene therapy strategy for treating corneal disease. In the first year study, we found that the activity of k12 promoter increased to a constant level from 1.0 to 2.5 kb by in vivo Gene Gun delivery transfection. This result encourageed us to investigate the more detailed structure with k12 promoter. At first we focused on the Pax-6 pair boxes located within 2.5 KZ. There are four Pax-6 pair boxes located upstream from the transcriptional initiation site to 2.5 kb. We construct several deletion constructs, including 2.0 KZ / 0.2 KZ, 2.0 KZ /0.4 KZ, and 2.0 KZ /0.6 KZ deletion of Pax-6 elements, and we found that the promoter activities decreased when the number of Pax elements decreased. It suggests that the Pax pair boxes in the promoter of k12 did play important roles in the regulation of k12 expression. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/26704 | 其他識別: | 882314B002372 | Rights: | 國立臺灣大學醫學院眼科 |
顯示於: | 醫學系 |
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882314B002372.pdf | 61.32 kB | Adobe PDF | 檢視/開啟 |
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