發展四環黴素誘發基因表現及抑制系統

Abstract

利用轉殖基因表現或抑制來研究基因功能，以了解當細胞表現過多或缺乏某一功能性基因時會造成細胞或動物何種生理影響。本論文研製之四環黴素可調節系統(tetracycline inducible system)是目前廣泛使用於調節基因表現的系統之一，當添加四環黴素時即可誘發基因表現；利用四環黴素可調節式基因表現系統需要兩組質體DNA，分別含有調節性DNA片段及反應性DNA片段。本研究已構築只需ㄧ個DNA質體就可具有四環黴素可調節式基因表現，只需添加1 ng/mL於培養液中即可具有誘發基因表現之效，遠低於目前常用劑量500至1000倍。本系統亦證明在調節性DNA片段中控制四環黴素轉錄調節蛋白質基因之啟動子強度會影響未誘發狀態時本系統基因表現基礎量。本系統除可應用於誘發基因表現於一般性細胞外，亦分別利用小鼠乳腺腫瘤病毒、鼠及豬胰島素基因啟動子控制四環黴素轉錄調節蛋白質基因，加以構築可應用於乳腺上皮細胞或胰臟β細胞之可調節式基因專一表現系統。這些結果證明我們已經構築具高誘發性及低基礎量調節式基因表現DNA載體，便利產製具有四環黴素可調節式基因轉殖動物。本研究亦建立可嚴密監控、可調節轉殖基因表現及抑制的系統，使轉殖基因的表現及抑制同時具有誘發調控性。所建立之可調節系統是利用四環黴素來同時雙向調節轉殖基因的表現及抑制。本系統主要原件為U6啟動子、CMV啟動子、四環黴素活化蛋白質DNA結合片段及四環黴素活化蛋白質。本系統利用表現轉殖GFP綠螢光蛋白及抑制功能性mst3基因來加以驗證調控效果，由結果顯示本系統確實具有誘發物添加劑量效應及處理時間效應，達到嚴密監控、可調節轉殖基因表現及抑制之效果。用以產生shRNA之合成DNA能利用限制酶切接方式選殖入本系統載體DNA內，使本系統具有方便選殖及嚴密調控於抑制基因表現之優點。

Temporal control of the expression of transgenes or the selective knockdown of endogenous genes by the tetracycline (Tet)-induced transactivator has been broadly used in the studies of the functions of targeted genes in a cell or in an animal model. A conventional two-vector Tet-inducible expression systems exhibit several problems, such as time-consuming, costly, labor-intensive and low efficiency. Hence, the single-vector Tet-on systems were developed in this study to try to solve these problems. The developed single-vector Tet-on systems exhibited the high sensitivity to the induction of doxycycline (Dox) at the concentration as low as 1 ng/mL, which is 500 to 1000-fold lower than that usually used in other Tet-on systems. Further studies show that the basal activity of Tet-on systems depends largely on the strength of promoter that controls transactivator. In addition, the developed single-vector Tet-on systems could facilitate the generation of conditional transgenic cells and animals with minimum adverse effects from long-term administration of Dox. The tissue- or cell specific expression of transgene was also demonstrated by the Tet-based inducible vectors, whose transactivator is controlled by mouse mammary tumor virus (MMTV), the rat insulin II gene promoter (RIP) or porcine insulin gene promoter (PIP). The bipartite Tet-inducible systems for simultaneously expression of transgene and shRNA were also developed. In the developed bipartite Tet-inducible systems the tetracycline operator sequences (tetO) were placed upstream both the minimal CMV and minimal RNA polymerase II and U6 shRNA promoters. This bipartite inducible system allows to induce transgene expression and to suppress the target gene simultaneously. The versatility of the developed bipartite Tet-inducible systems was demonstrated by simultaneously inducing the expression of EGFP as well as the selective knockdown of mammalian Ste20-like protein kinase 3 (Mst3). Furthermore, the developed bipartite Tet-inducible systems could be tightly regulated by Dox in a concentration- and time-dependent manner.