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|標題:||Effect of Tet-controlled RNAi system on the gene expression in Drosophila Schneider 2 (S2) cell line
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Expression of highly controllable genes in insect cells using a modified tetracycline-regulated gene expression system. J Biotechnol. 80: 75-83. Yang, D., H. Lu, and J. W. Erickson. 2000. Evidence that processed small dsRNAs may mediate sequence-specific mRNA degradation during RNAi in Drosophila embryos. Curr Biol. 10: 1191-1200.||摘要:||
利用四環黴素調控系統（tetracycline-responsive expression system, TRES）表現以東方果實蠅（Bactrocera dorsalis (Hendel)）肌動蛋白（actin）基因所設計的短髮夾核醣核酸（small hairpin RNA, shRNA）片段於果蠅Drosophlia Schneider 2 (S2) 細胞株，並測試TRES調控shRNA之表現及其對細胞之影響。本試驗構築之四環黴素調控系統，係以驅動（driver）載體上之東方果實蠅卵黃蛋白（yolk protein, yp）基因啟動子驅動四環黴素轉錄活化子（tetracycline-controllable transactivator, tTA）表現，再經由此tTA與表現（responder）載體上四環黴素操作子（tet operator）鍵結而促進後方shRNA片段表現；然而，tTA之活性可因加入四環黴素而被抑制，使之無法與其操作子結合而終止shRNA表現。卵黃蛋白啟動子僅於雌成蟲發育卵時受激素調控而活化，在S2細胞測試結果顯示此啟動子能被濃度為10-7-10-9 M 的20-羥基蛻皮激素（20-hydroxyecdysone, 20E）激活而表現tta 基因。然而，濃度高於10-7 M之20E雖可刺激tta大量表現，但細胞存活數卻明顯降低；此外，經雙轉染（co-transfection）驅動及表現載體之細胞，以10-8 M之20E處理後，可以偵測到shRNA片段的表現，顯示tTA與操作子結合後確實能轉錄出shRNA，顯示此系統在S2細胞中可以發揮作用，期望不久的未來能將此系統有效的應用於東方果實蠅蟲體內。
In this study, a tetracycline-responsive expression system (TRES) for expressing a small hairpin RNA (shRNA), i.e. part of actin gene of Bactrocera dorsalis (Hendel), was modified in Drosophila S2 cell line. This could be applied as an alternative method to control insect pests. For the driver construct of TRES, the promoter of yolk protein gene (yp) of B. dorsalis, was selected to drive the expression of tetracycline-controllable transactivator (tTA); and the expressed tTA proteins then bond to the tet operator on the responder construct of TRES to initiate the transcription of the DNA coding shRNA sequence; furthermore, the tTA activity could be inhibited by adding tetracycline, and under this circumstances, the shRNA expression was stopped. The yp promoter was only activated by hormone when the eggs started to develop in female adults, and proved to be activated under the stimulation of 10-7-10-9 M 20-hydroxyecdysone (20E) in the driver-transfected S2 cells. In the driver-transfected S2 cells, tta was significantly expressed by treating with 10-7 M, resulting in a large amount of cell death. In the cells co-transfected with both driver and responder, the shRNA expression was clearly detected after treating with 10-8 M 20E, suggesting that the tTA protein is able to activate the shRNA expression in S2 cells. In conclusion, it is demonstrated that this TRES is functional in S2 cells, and that, in the near future this system can further transferred into B. dorsalis to observe whether the system can function as well in this fly.
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