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研究生:王乃玉
研究生(外文):Nai-Yu Wang
論文名稱:設計方法以濃縮取得特定選擇性剪接所產生之mRNAisoform並進行ADAR2bisoform的選殖
論文名稱(外文):Methods for Enrichment of an mRNA Isoform with Specific Alternative Splicing and Cloning of ADAR2b Isoform
指導教授:歐樂君
指導教授(外文):Lo-Chun Au
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:英文
論文頁數:67
中文關鍵詞:選擇性剪接增殖腺核苷解胺酶
外文關鍵詞:alternative splicingenrichmentADAR2isoform
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在高等真核生物當中,pre-mRNA上的選擇性剪接(Alternative splicing)是調控基因表現的一種重要機制。近年來,有許多針對選擇性剪接所進行的基因體分析,顯示人類約有40~60%的基因會因為受到選擇性剪接而產生不同形式(isoform)的mRNA。雖然這些isoform 是以相對上較少的數量存在,但也因為這些isoform是由不同排列組合所構成,因而造就了不同功能蛋白質的產生。為了深入了解不同isoform可能產生的蛋白質功能,對應mRNA的選殖(cloning)與表現是必要的過程。在這裡,我們想要在反轉錄-聚合酶連鎖反應(RT-PCR)及選殖之前設計方法來放大欲得之mRNA isofirm。負選擇(negative selection)方法是用來破壞欲排除的mRNA variants,相反地,正選擇(Positive selection)方法則是釣取欲得之mRNA isoform。我們以大鼠的腺核苷解氨酶二(ADAR2)之mRNA作為實驗材料,發現以負選擇方法及正選擇方法分別可以提高欲得isoform達到16倍及19倍的放大效果。除此之外,負選擇方法與正選擇方法不論是獨立運用或聯合運用,都可以促進微量mRNA isoform的分離與選殖。再者,利用這些方法也可以幫助我們了解pre-mRNA上所發生選擇性剪接事件(Alternative splicing events)之間的關聯性。另一方面,由於到目前為止,ADAR2作用基質的發現仍然十分有限,我們想利用已選殖並轉殖(transfection)成功的ADAR2b,在細胞內做大量表現(Over-expression)。這套表現系統也許有利於將來找尋ADAR可能的作用基質。
Alternative splicing of pre-mRNA is an important mechanism for regulating gene expression in higher eukaryotes. Recent genome-wide analyses of alternative splicing indicated that 40~60 % of human genes have alternatively spliced isoforms, although some variants exist only in relatively low abundance. It has been shown that proteins of different functions can be produced by a diverse array of mRNA derived from a single pre-mRNA. Inevitably cloning and expression of the corresponding mRNAs (cDNA) constitute an essential step toward understanding the function of such protein isoforms. Here we propose methods for enriching an mRNA isoform, which is carried out before the RNA preparation is subjected to RT-PCR and cloning. While the negative selection method destroys unwanted mRNA variants, the positive selection enriches the desired one. Focusing on the mRNA of rat ADAR2 (adenosine deaminase 2 that acts on RNA) as an example, we have achieved 16 folds and 19 folds of enrichment of the given mRNA variant via the proposed negative and positive selection respectively. Single use or combined uses of our method facilitate the isolation and cloning of a minor mRNA variant. Moreover, these methods can also be used to determine whether the two alternative splicing events taking place in a pre-mRNA are linked.
Since only a limited number of ADAR2 substrates were discovered so far, by transfecting cells with successfully cloned ADAR2b, we will attempt to find the potential substrates of ADAR2 through overexpression in the future.
致 謝 1
中文摘要 2
Abstract 3
Table of Contents 4
List of Tables 6
List of Figures 7
List of Abbreviations 8

Chapter 1: Introduction 9
1.1 RNA splicing 9
A. An overview of RNA splicing: 9
B. The importance of alternative splicing: 9
C. The splicing signals: 10
D. The splicing mechanisms: 10
1.2 RNA editing 13
A. Overview: 13
B. Two distinct mechanisms of RNA editing: 13
a. Insertion/Deletion Editing: 13
b. Substitution Editing: 14
1.3 ADAR 16
A. Overview 16
B. The ADAR family 19
C. The common domain structure of ADAR 19
D. ADAR specificity 21
1.4 ADAR2 22

Chapter 2: Objectives 27

Chapter 3: Materials and Methods 28
3.1 Cloning of active form ADAR2 (ADAR2b) 28
A. Total RNA extraction from tissue 28
B. Negative selection 28
C. Positive selection 30
D. Reverse Transcription (RT) 31
E. Polymerase Chain Reaction (PCR) 31
F. Agarose Gel Electrophoresis 31
G. DNA sequencing 32
3.2 Regulation of ADAR2 expression by Tet-off system in cells 32
A. Construction of ADAR2 plasmid 32
a. PCR 32
b. Restriction enzyme cutting and DNA sequencing 32
c. Ligation 33
B. To harvest the ADAR2-expression plasmids 33
a. Transformation 33
b. Quick screen for colonies 35
c. Purification and identification of the plasmids from colonies 35
d. Amplification of ADAR2 plasmid 36
C. Expression of ADAR2 in cells 37
a. Cell culture 37
b. Stable transfection: 37
c. Induction of ADAR2 and extract total RNA from cells 38
d. RT-PCR for ADAR2 38
e. Protein extraction from cells/tissues 39
f. Protein concentration measurement 40
g. Western blot analysis for ADAR2 40
3.3 To find the potential substrates of ADAR2 43
A. Test the efficiency of universal primers 43

Chapter 4: Results 45
4.1 Developing a method for enriching specific mRNA isoform of ADAR2 45
A. Enrichment of –47nt isoform by negative selection and positive selection 45
B. Enrich the isoform of ADAR2b 50
C. The linkage between two individual alternative splicing events 52
4.2 Over-expression of ADAR2 in CHO cells 52
A. Induction of ADAR2 by Tet-off regulatory system 52
B. The RNA level of ADAR2 expression 52
C. The protein level of ADAR2 expression 54
4.3 Find the potential substrates of ADAR2 54
A. Design the universal primers 54

Chapter 5: Discussion 59
Reference 62
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