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研究生:陳茗蓉
研究生(外文):Ming-Jung Chen
論文名稱:建立並分析表現不同片段長度Tcap之轉基因斑馬魚
論文名稱(外文):Establishment and characterization of transgenic zebrafish lines expressing truncated forms of Tcap
指導教授:黃銓珍
指導教授(外文):Chang-Jen Huang
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:英文
論文頁數:43
中文關鍵詞:斑馬魚肌肉萎縮症肢帶型肌肉萎縮症2G型
外文關鍵詞:TcapLGMD 2Gzebrafishmuscle dystrophy
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Tcap,在人類稱為Telethonin,為在肌肉上Z-disc 表現的蛋白質,主要表現在骨骼肌與心肌。Tcap 的功能尚未清楚,目前研究認為Tcap會與其他Z-disc 上的蛋白質例如:titin、MLP、minK 等進行連結,並對肌肉的構造和訊息傳遞扮演重要的角色。目前已知tcap 是造成人類肢帶型肌肉萎縮症2G 型(LGMD 2G)的致病基因,但致病機制仍不清楚。
本研究主要在建立並分析表現Tcap truncated form 之轉基因斑馬魚。根據Tcap 與其他蛋白質的結合區,我們建立了不同的truncated form 之Tcap,並將其表現在斑馬魚並且使其變成穩定基因轉殖魚,藉由loss of function 分析Tcap 與其他蛋白質交互作用之重要性。
本研究藉由骨骼肌專一性啟動子α-actin promoter 將不同truncated form 之Tcap 表現於骨骼肌 ,透過共軛焦顯微鏡影像確定其表現於骨骼肌的Z-disc 上,之後建立不同truncated form 的基因轉殖魚,並且命名為R1、R2、R3、F2。正常斑馬魚在2-3 天時即會分泌破殼酵素,伴隨著擺動身軀以破殼孵化。在本研究中,發現在這些穩定轉基因魚孵化率出現了異常,原因可能為肌肉上有缺陷造成無法順利破殼,因此透過共軛焦影像來觀察肌肉的型態,並記錄其泳動能力。另一方面,我們利用免疫染色來觀察肌肉的結構是否受損。
藉由本研究,希望能瞭解Tcap 在LGMD 2G 疾病中扮演的角色與致病機制。未來對於LGMD 2G 的治療給予一些幫助。
Tcap (also termed telethonin, titin-cap), a 19-kDa protein with a unique β-sheet structure, is exclusively expressed within the Z-disc of adult skeletal and cardiac muscle. Tcap provides binding sites for titin and other Z-disc-associated proteins, and it has also been found that these binding sites of Tcap are lost in the patients of limb-girdle muscular dystrophy type 2G (LGMD 2G). However, the mechanism that Tcap deficiency causes LGMD 2G is currently unknown. Hence, Tcap may be an important therapeutic target in the treatment of patients with limb girdle muscular dystrophy.
In this study, we have established and analyzed the transgenic zebrafish lines expressing various truncated forms of Tcap. On the other hand, we have characterized their skeletal muscle phenotype. The results showed that the abnormality of muscle fibers was definitely observed in
transgenic line Tcap-R3, which lacks the docking sites for titin and other Z-disc-associated proteins. Furthermore, the sarcomere assembly was not affected in Tcap transgenic lines using IHC staining. On the other hand, we also analyzed the hatchability and swimming ability of these transgenic lines at 3 dpf.
In present study, we established transgenic zebrafish lines expressing truncated forms of Tcap, and further provided a novel animal model for studying the disease-causing echanism of LGMD 2G.
Acknowledgements..........................................i
中文摘要..................................................ii
Abstract................................................iii
Contents.................................................iv
Contents of Table.........................................v
Contents of Figure.......................................vi
Introduction..............................................1
1. Structure of skeletal muscle and muscle development in the zebrafish ........................................... 1
2. The sarcomeric Z-disc and Z-disc protein ................................................. 2
3. Tcap.................................................. 3
4. Muscular dystrophies and LGMD 2G ..................... 5
5. Zebrafish as a model for muscular dystrophy .......... 7
Specific aims ........................................... 9
Materials and Methods .................................. 10
Zebrafish .............................................. 10
Polymerase chain reaction .............................. 10
Agarose gel electrophoresis ............................ 11
DNA ligation ........................................... 11
Competent cells preparation .............................11
Transformation ......................................... 12
Restriction endonuclease digestion ..................... 12
Microinjection and established transgenic line ......... 12
Preparation of Genomic DNA ............................. 13
Statistical analysis ................................... 13
Whole mount Immunostaining ............................. 14
Phalloidin staining .................................... 14
Evans Blue dye (EBD) labeling........................... 15
Results ................................................ 16
1. Establishment of transgenic zebrafish lines expressing truncated forms of Tcap..................................16
2. Transgenic line Tcap-R3 showed similar phenotypes of Muscular dystrophy ... ..................................18
3. Immunostaining of transgenic zebrafish with components of muscle structure......................................19
Discussion ............................................. 22
References ............................................. 26
Tables and Figures ..................................... 34
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