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標題: | 利用tetracycline regulatory system 表現 cardiac Troponin C Using tetracyc1ine-controlled transcriptiona1 system to express zebrafish cardiac troponin C |
作者: | Wei-Yuan Tsai 蔡維原 |
出版年 : | 2003 |
學位: | 碩士 |
摘要: | Tet System 是由 Gossen 跟Bujard 在 Escherichia coli發現的 transcriptional repression system 所發展而來,藉由 tetracycline 跟 tTA ( transcriptional repressor )結合後,可抑制欲研究基因的表現。而後 Gossen 等人又將 tTA 結合VP16 ( transcriptional activator)而成 rtTA ,使得在 tetracycline 存在的環境下,rtTA 會跟 tetracycline 結合並開敔特定基因的表現 ( tet-on system )。在我們的實驗中,利用 tet-on system 配合斑馬魚心臟專一表現基因( cardiac myosin light chain )?動子,在斑馬魚心臟專一表現 cardiac Troponin C(cTnC)。cTnC 是一個鈣離子結合蛋白,在cTnC 和鈣離子結合後,actin可以跟 myosin 結合使得肌纖維產生收縮。我們架構了一個心臟專一表現的雙向?動子,用以同時驅動EGFP報導基因以及 cTnC-antisense 在心臟專一表現,利用 antisense 抑制內生性基因表現,來研究 cTnC 的基因功能。 在基因轉殖魚胚胎發育到 12 小時,添加入 tetracycline 衍生物 doxycycline ,可以在 12 小時後看到綠色螢光在心臟專一表現。在 48 小時大的魚,可以觀察到 cTnC-antisense 的心跳速率顯著的較控制組來的快( cTnC – antisense l66.8 每分鐘;控制組 137.6 每分鐘) ; 但是在 6 天大的魚中, cTnC-antisense 的心跳數卻是明顯的慢於控制組(cTnC-antisense 190.9 每分鐘;控制組219.5 每分鐘)。當進行 cTnC morpholino 注射實驗時,在 1.15ng 劑量注射過的魚中,會出現心室不會跳動的性狀;而在 0.575ng 劑量注射的魚中,並未出現心室停止跳動的性狀,跟控制組比較起來,心跳數有些微變快的性狀。藉由 cTnC-antisense 所產生心臟異常性狀跟藉由較低劑量 cTnC morpho1ino 所產生的變異相似。藉由這樣的證據,可以支援藉由 tetracycline regulatory system 所驅動的可調控的基因表現系統在斑馬魚中可以使用,同時也提供另一種研究特定基因的實驗方式。 The tetracycline-controlled transcription system provides a convenient method to study stage-depended gene expression. Here, we report an inducible tet-on system to study the zebrafish cardiac troponin C (cTnC), a calcium binding protein. We constructed a bi-directional vector, in which GFP and antisense of cTnC were driven by a cardiac specific promoter, cmlc2. When 12-hpf embryos were treated with 1 μg/ml doxycycline, we found that GFP was specifically expressed in the hearts of embryos after induction for 12 hr. We also found that the hearts were slightly bigger than that of wild-type embryos and the heart-beating rate was little faster than wild-type (166.8 vs 137.6 per minutes). When cTnC-morpholino (cTnC-MO) was injected into one-celled fertilized eggs, the 1.15ng-injected embryos showed a silent ventricle syndrome. No silent ventricle occurred in the 0.575ng-injected embryos and instead the heart —beating rates were slightly faster than those of wild-type. The heart defects were caused by cTnC knock-down due to cTnC-antisense induction was similar to those caused by the lower dosage treatment of cTnC-MO. These evidences strongly suggest that this inducible system may work on model fish so that it provides an approach to study gene function at any desired developmental stages. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75433 |
全文授權: | 未授權 |
顯示於系所單位: | 漁業科學研究所 |
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