Title page for 972204025


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Student Number 972204025
Author Ching-Wen Su(蘇靖雯)
Author's Email Address No Public.
Statistics This thesis had been viewed 714 times. Download 911 times.
Department Life Science
Year 2010
Semester 1
Degree Master
Type of Document Master's Thesis
Language zh-TW.Big5 Chinese
Title Delineating the expression pattern of FoxO6 in mouse skeletal muscle
Date of Defense 2011-01-05
Page Count 79
Keyword
  • FoxO6 antibody
  • FoxO6 expression pattern
  • skeletal muscle
  • Abstract Forkhead transcription factors constitute a family that share a conserved DNA binding domain, the Forkhead box. Members of the subfamily O, including FoxO1, FoxO3, FoxO4, and FoxO6, are involved in many physiological processes, such as cell cycle arrest, DNA damage repair, cell differentiation and apoptosis. Among these FoxOs, the expression and function of the recently discovered FoxO6 in most physiological processes has not been studied before. Therefore, we want to delineate the expression of FoxO6 at mRNA and protein levels respectively in developing muscles in utero and in various post-natal mature muscles. So we detected FoxO6 mRNA in mouse tissues by Q-PCR and observed that the heart, liver, kidney have lower FoxO6 expression than brain and muscle. The highest expression of FoxO6 was found in pituitary. We also found that FoxO6 expression decreased with increasing age. It is also of interest to know the Foxo6 localization at the different stages of myoblasts. To achieve these goals, we used FoxO6-speific domain (amino acid 229~492) as antigen to produce FoxO6 polyclonal antibody in rabbits. After titering, we are sure that this antibody can recognize FoxO6 specifically without cross-reacting with other FoxOs. Using immunofluorescence, we demonstrated that FoxO6 accumulated mostly in nucleus of proliferating myoblasts, and this nuclear localization was further increased when myoblasts became confluent. After terminal differentiation, homogeneous distribution of FoxO6 was detected in the cytoplasm and nucleus of myotubes. Since the expression pattern of FoxO6 is similar to other FoxOs, it suggests that the role played by FoxO6 in myogenesis is probably the same as other FoxOs do. After the delineation of its expression, we will perform promoter assay to find out how FoxO6 expression is regulated in muscle.
    Table of Content 目錄
    中文摘要‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ⅰ
    英文摘要‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ⅱ
    誌謝‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ⅲ
    目錄‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ⅳ
    圖目錄‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ⅷ
    第一章、緒論 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥1
    1.肌肉的發育‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥1
    2.肌肉構造‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥2
    3.FoxO轉錄因子 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥4
    4.研究動機與目的‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥7
    第二章、實驗方法與材料 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥9
    2.1.實驗材料‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥9
    2.1.1菌株
    2.1.2菌株培養
    2.1.3菌株保存
    2.1.4小鼠組織
    2.1.5肌肉組織冷凍包埋
    2.1.6抗體宿主
    2.1.7細胞株與細胞培養
    2.2.質體建構 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥10
    2.2.1pcDNA3.0-mFoxO6表現載體的製備
    2.2.2pMAL-C2X-mFoxO6 +685~+1476 表現載體的構築
    2.2.3pET-32a-mut-mFoxO6+685~+1476表現載體的構築
    2.2.4pcDNA 3.1-mFoxO3/4表現載體的製備
    2.3.RT-PCR ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥12
    2.3.1Total RNA的抽取 ( RNA extraction )
    2.3.2反轉錄酶反應 ( Reverse Transcription, RT )
    2.3.3聚合酶鏈反應 ( Polymerase Chain Reaction, PCR )
    2.3.4Real-time PCR ( Q-PCR )
    2.4.mFOXO6的抗體製備 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥13
    2.4.1誘導 mFOXO6 抗原蛋白表現
    2.4.2mFOXO6抗原蛋白質的純化
    2.4.3誘導宿主免疫反應
    2.5.西方墨點實驗 ( Western blot ) ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥16
    2.5.1總蛋白質的製備
    2.5.2SDS-polyacryamide Gel Electropheresis
    2.5.3轉印 ( Transfer )
    2.5.4Blocking以及Antibody辨識
    2.5.5抗體脫附 ( Striping )
    2.6.冷凍切片‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥18
    2.6.1載玻片的表面處理
    2.6.2冷凍切片機上機流程
    2.7.組織化學染色法 ( immunohistochemistry ) ‥‥‥‥‥‥‥‥‥‥‥‥18
    2.7.1H&E stain
    2.7.2ATPase stain
    2.7.3免疫螢光染色 ( immunofluorescence )
    2.8.TNT in vitro translation‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥21
    第三章、結果‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥22
    3.1.mFoxO6抗原的製備與純化 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥22
    3.2.抗體的誘發與測試 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥24
    3.3.偵測細胞株總蛋白質中 FoxO6 的表現 ‥‥‥‥‥‥‥‥‥‥‥‥‥25
    3.4.肌纖維母細胞在不同時期的 FoxO6 localization‥‥‥‥‥‥‥‥‥‥26
    3.5.在小鼠後腿肌肉中 FoxO6 的 mRNA 表現位置和快縮肌/慢縮肌的關連 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥27
    3.6.在小鼠組織中 FoxO6 的 mRNA 表現情形 ‥‥‥‥‥‥‥‥‥‥‥28
    第四章、討論‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥29
    4.1.在 C2C12 中 FoxO6 對肌肉分化的影響及其作用‥‥‥‥‥‥‥‥‥29
    4.2.FoxO6在 C2C12 中的表現位置對其分化情形的影響 ‥‥‥‥‥‥‥29
    4.3.年紀對 FoxO6 表現情形的影響 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥30
    4.4.探討 FoxO6和肌纖維中快縮肌與慢縮肌的關連性‥‥‥‥‥‥‥‥‥31
    4.5.未來要進行的實驗 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥31
    第五章、參考文獻‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥33
    第六章、圖表‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥35
    附錄一 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥68
    I.試劑配方 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥68
    II.酵素和限制酶 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥69
    III.抗體‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥70
    IV.藥品試劑 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥70
    附錄二 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥71
    I.Primer對照表 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥71
    II.Plasmid對照表‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥71
    III.Q-PCR primer對照表‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥72
    附錄三 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥73
    藥品縮寫及全名‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥73
    附錄四 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥74
    實驗方法與材料之表格‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥74
    附錄五 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥76
    質體建構之基本流程‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥76

    圖目錄
    圖一、老鼠的 FoxO 家族成員其 coding region ( CDS ) 及蛋白質的序列比較 …‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥35
    圖二、利用 MBP fusion protein system 將抗原蛋白表現出來‥‥‥‥‥‥‥‥37
    圖三、pET protein purification system‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥40
    圖四、pET-32a-mut-mFoxO6+685~+1476的質體建構‥‥‥‥‥‥‥‥‥‥‥43
    圖五、製作His-tagged Foxo6抗原蛋白‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥45
    圖六、利用不同濃度的eluent來確認所沖提出來的蛋白質確實含有His-tagged‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥47
    圖七、兔子免疫前血漿及免疫一個月後之血漿測試 ‥‥‥‥‥‥‥‥‥‥‥48
    圖八、兔子免疫兩個月後之血清測試‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥51
    圖九、確認抗體對 FoxO family 的辨認專一性 ‥‥‥‥‥‥‥‥‥‥‥‥‥53
    圖十、肌纖維母細胞 ( C2C12 ) 中的 FoxO6 蛋白質表現量‥‥‥‥‥‥‥‥55
    圖十一、在 HEK 293T 中過量表現 mFoxO6 後其蛋白質表現情形 ‥‥‥‥56
    圖十二、C2C12 parental 在不同時期的 FoxO6 localization‥‥‥‥‥‥‥‥‥57
    圖十三、pcDNA3.0-mFoxO6 +685~+1474 質體建構 ‥‥‥‥‥‥‥‥‥‥‥61
    圖十四、原位雜交的探針製備‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥63
    圖十五、以組織化學染色法觀察十二個月小鼠後腿肌肉 ( GC ) 的冷凍連續切片 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥64
    圖十六、比較在六個月的小鼠不同組織間 FoxO6 mRNA 表現情形‥‥‥‥‥65
    圖十七、比較不同年紀的小鼠組織中 FoxO6 mRNA 表現情形‥‥‥‥‥‥‥66
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    Advisor
  • Shen-Liang Chen(陳盛良)
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    Date of Submission 2011-01-19

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