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研究生:黃鼎皓
研究生(外文):Ting-Hao Huang
論文名稱:分析Nolz-1基因上游基因調控區域以及構築Nolz-1基因條件式基因剔除小鼠之基因標的載體
論文名稱(外文):Analysis of the upstream regulatory elements of Nolz-1 gene and construction of the conditional gene targeting construct of Nolz-1 gene
指導教授:劉福清劉福清引用關係
指導教授(外文):Fu-Chin Liu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:神經科學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:67
中文關鍵詞:紋狀體
外文關鍵詞:striatum
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紋狀體是在基底核中主要的訊號接收核區,並認為參與跟許多腦功能,例如意志的運動控制、認知上的功能以及回饋學習與控制有關。 若紋狀體的功能有缺失會造成許多的神經疾病,如帕金森氏症 (Parkinson’s disease)、杭亭頓舞蹈症 (Huntington’s disease)、強迫症以及藥物成癮。 為了瞭解紋狀體的發育,本實驗室找到了一個大量表現在紋狀體發育時期的結構,lateral ganglionic eminence (LGE),的新穎基因,Nolz-1。 本篇論文中為暸解控制Nolz-1 表現的機制,我利用不同長度的Nolz-1 5'-flanking regions 接上luciferase reporter gene,轉染至Neuro2A 細胞和ST14A 細胞,以分析promoter 的活性。 發現再轉譯起始子 (start codon,ATG) 上游2.4 kb 之間的片段之內在Neuro2A 以及ST14A 都有著相似的promoter 活性趨勢。 5’-flanking region 從 -0.2 kb 到 -0.8 kb 中間promoter 活性慢慢地上升,5’-flanking region 從 -0.8 kb 到 -1.1 kb 以及 -1.5 kb 到 -1.6 kb 這兩個片段有增強活性的效果,而5’-flanking region 從 -1.1 kb 到 -1.5 kb 和 -1.6 kb 到 -2.4 kb 這兩段則有抑制活性的效果。 另外,5’-flanking region 從 -2.4 kb 到 -6.7 kb 之間在ST14A 細胞中有專一性的活性增強現象。 我們也試著分析調控Nolz-1 在神經分化過程中,promoter 被活化的區域。 我們利用aggregation 外加retinoic acid 促使P19 細胞進行神經分化後,觀察Nolz-1 不同5’ 端上游片段的活性變化,發現在5’-flanking region 從 -0.2 kb 到 -0.45 kb 中的片段就有promoter 活性增強的效果。 另外一方面,利用比對不同物種間Nolz-1 同源基因的基因組,我發現不同物種Nolz-1 的3’-UTR 有高度相似性。 將這相似的3’-UTR 片段接入reporter gene 的3’-UTR 的位置,發現保守的3’-UTR 片段會降低reporter gene 的mRNA 以及蛋白質表現量。 綜合這些研究我們可以初步了解調控Nolz-1 表現的區域位置。 最後因為計畫能進一歩了解Nolz-1 在紋狀體發育過程中的功能,所以利用recombineering-based 的技術構築條件式基因剔除小鼠的基因標的質體,希望日後Nolz-1 條件式基因剔除小鼠產生後,可以藉著與不同的Cre 基因轉殖小鼠交配,觀察Nolz-1 功能喪失的情況下,神經系統發育的變化,而藉以瞭解Nolz-1 基因的生物功能。
The striatum is the major recipient of inputs to the basal ganglia from the cerebral cortex. The striatum is known to be involved in processing multiple neurological functions, like the planning and modulation of movement, cognitive processes, and the reward learning. The malfunctions of striatum are involved in the pathogenesis of several neurological diseases, including Parkinson’s disease, Huntington’s disease, obsessive-compulsive disorder, and drugs addiction. In order to understand the development of such important structure, our laboratory has previously identified a novel gene, Nolz-1, that expressed in the striatal anlage, the lateral ganglionic eminence (LGE). In this study, I analyzed a series of luciferase reporter gene constructs containing different lengths of the 5’-flanking regions of Nolz-1 upstream of the translation initiation site. Reporter constructs were transiently transfected into Neuro2A and ST14A cells to test the cell type specificity of the promoter activity. Transient transfection of the series of reporter gene constructs showed that, in both Neuro2A and ST14A cells, the constructs containing -2.4 kb region displayed similar pattern of promoter activity. Transfection of the constructs containing 5’-flanking region between -0.2 kb and -0.8 kb showed gradually increased promoter activities. The two fragments from -0.8 kb to -1.1 kb and from -1.5 kb to -1.6 kb enhanced the promoter activity; however, the other two regions from -1.1 kb to -1.5 kb and -1.6 kb to -2.4 kb repressed the promoter activity. Moreover, there is a specificity of increased promoter activity in ST14A cells with the 5’-flanking region between -2.4 kb and -6.7 kb. I also tried to test which 5’-flanking region regulated the promoter activity that was involved in Nolz-1 expression during neuronal differentiation. I transfected a series of deletion 5’-flanking region constructs into P19 cells, and studied the changes of the promoter activity during neural induction by aggregation and retinoic acid treatment. I found that there is a significant enhancement of promoter activity after neural induction in the region between -0.2 kb and -0.45 kb. In order to identify evolutionally conserved DNA sequences that regulate Nolz-1, I compared the genomic sequences of the homologs of Nolz-1 among different species, and found that the 3’-UTR of Nolz-1 was highly conserved between vertebrate animals. To characterize the conserved 3’-UTR function, I inserted the conserved 3’-UTR into the 3’-UTR region of luciferase gene. Transfection this construct into Neuro2A cells showed that the conserved 3’-UTR reduced the expression of the reporter gene at both mRNA and protein levels. Taken together, my studies provide an initial characterization of the regulatory regions in 5’- and 3’-flanking regions of Nolz-1 gene. In order to further study the roles of Nolz-1 during striatal development, I planned to generate Nolz-1 conditional knockout mice. Therefore, I successfully used the recombineering-based method to construct the Nolz-1 conditional gene targeting construct in which the exon3 of Nolz-1 were flanked by loxP sites. In the future, when Nolz-1 condition knockout mice are crossed with Cre transgenic mice under control of different tissue-specific promoters, we can analyze neural development in the mutant mice to elucidate the biological function of Nolz-1 gene.
中文摘要 -------------------------------------------------------------------------------------------- 1
英文摘要 -------------------------------------------------------------------------------------------- 3
緒論 -------------------------------------------------------------------------------------------------- 5
材料與方法 ----------------------------------------------------------------------------------------- 9
結果
一、 分析Nolz-1 上游調控區域 (cis- regulatory elements) ------------------------- 18
二、小鼠Nolz-1 條件式基因剔除載體之建構 --------------------------------------- 22
討論 ------------------------------------------------------------------------------------------------ 26
參考文獻 ------------------------------------------------------------------------------------------ 34
圖表 ------------------------------------------------------------------------------------------------ 46
附錄 ------------------------------------------------------------------------------------------------ 67
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