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研究生:呂史提
研究生(外文):Steve Leu
論文名稱:探討Pinin在鼠類胚胎發育之功能
論文名稱(外文):Characterization of the role of Pinin in mouse development
指導教授:歐陽品
指導教授(外文):Pin Ouyang
學位類別:博士
校院名稱:長庚大學
系所名稱:基礎醫學研究所
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:2005
畢業學年度:93
語文別:英文
中文關鍵詞:胚胎發育基因剔除啟動子小鼠
外文關鍵詞:pininmousepromoterdevelopmentknockout
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Pinin (pnn) 是一個同時存在於胞橋小體 (desmosome) 及核斑 (nuclear speckle) 的多功能蛋白質,我們決定製造出缺乏pinin的小鼠模式來探討它的生物功能。經過分析 pnn 在胚胎時期的表現形態,我們發現 pnn 在雙細胞時期的受精卵中就開始表現,同時它在各個胚胎發育時期皆有表現。此外在中樞神經系統以及E13.5時期後的表皮組織,也都有發現較高的pinin表現。而在分析 pnn 的基因結構後,我們發現pinin含有7080 個鹼基對 (bp) ,9個內插子 (exon),一個位於第八個外插子的SINE (short interspersed element),同時也發現第一個內插子被包埋在CpG islands 中。而藉由冷光活性分析 (luciferase activity assay) ,我們發現了位於轉錄起始點上游 180 至 200 鹼基對 (-200 to -180) 的起動子 (promoter) 區域在 pnn 的表現中扮演著重要的角色。而在與冷光活性分析結合的詳細定址實驗中,則是指出 -151 到 -148這個被轉譯因子GABP 所調控的 Ets 核心區域對於 pnn 的基本表現而言是必要的。而我們最後也製造出缺乏 pnn 的小鼠模式,而實驗結果也指出缺乏 pnn 會造成小鼠的早期胚胎死亡,而在 siRNA 的實驗中也證實了 pnn 對於細胞的存活的必要性。
Pnn is a moonlighting protein with dual location in different cellular compartments. To characterize its biological function, we aim to generate a mouse model deficient for pnn. Analysis of pnn expression in mouse embryos revealed pnn expression starts from the 2-cell fertilized egg stage and is ubiquitous at all stages of mouse embryo development. In addition, higher expressions of pnn in central nervous system and in skin after stage E13.5 was also be found. Gene structure analysis of pnn showed that mouse pnn is 7080 bp in length and contains 9 exons, one SINE in intron 8, and the first exon is embedded in CpG islands. Through luciferase activity assay and EMSA, we found that the region -200 to -180 in the pnn promoter plays an important role in regulation of pnn expression. Detailed mapping experiments combined with the luciferase reporter assay suggested that Ets core motif (-151 to -148) is essential for basal expression of pnn and the transcription factor GABP is involved in this regulation. Finally, we generated a mouse model deficiency in pnn, and the results showed that deficiency in pnn leads to early embryonic lethality. The siRNA experiment also indicated that pnn is essential for cell survival
Abstract  2
Introduction  3
Chapter I
Spatial and temporal expression profile of Pinin during mouse development

Abstract 
Introduction 
Results and Discussion 
Materials and Methods 

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Chapter II
Gene structure and promoter analysis of mouse Pinin
Abstract 
Introduction 
Results and Discussion 
Materials and Methods 

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Chapter III
Targeted disruption of Pnn results in mouse early embryonic lethality

Abstract 
Introduction 
Results and Discussion 
Materials and Methods 

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Summary  58
References  62
Figures Legends  73
Figures  84
Tables  104
Appendix  107
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