應用條件化基因剔除及轉基因策略探討小鼠C型血小板生長因子(PDGF-C)生理作用之分子調控機制(2/3)

近年來，多種可用於生物體外且具備可誘導性質之基因表現系統陸續發展應用，遂得以進一步突破傳統產製轉基因動物之諸多限制，而能逕行經由四環黴素調控系統表現系統之應用，詳實探討特定基因在生物體內扮演之生理功能。基於前述事實，本研究首要目標，乃積極嘗試使用四環黴素來控制rtTA 的表現;試圖將PDGF-C 結合於rtTA 相對應之啟動子下游，俾可藉由四環黴素有效控制PDGF-C 基因之表現 (tet operatorsequence; tet O)，達成條件化控制目標蛋白質(PDGF-C)之轉錄作用;且能夠進一步讓四環黴素辨識及調節轉C 型血小板生長因子基因轉殖鼠之表現與否，從而有效監控轉殖基因之表現量。鑑於內源性PDGF-C 基因之表現係受特定活化之啟動子(promoter)所控制，設若令該活化啟動子置換成為可接受四環黴素控制支配的轉錄活化子(transactivator) tTA 及rtTA 來調控，將使同時具備該二種構築的轉基因小鼠，可藉由使用四環黴素來調控目標基因的生理作用。基於此一原理，本研究首先成功構築一表現載體(Expression vector)及二種反應載體(Response vector)包括攜帶有特定專一性表現之啟動子諸如:四環黴素反應啟動子(Tet-responsive promoter)，與同時攜帶有報導基因(綠螢光蛋白質)及本研究擬深入探討之PDGF-C 目標轉殖基因。 初步結果已知該表現載體與反應載體混合雙基因顯微注射（co-injection）於小鼠原核階段胚，共係有tet-on 和 tg pBi-EGFP-PDGFC 與tet-on 及Tg pTRE2hyg-PDGFC 兩組;其中前者合計生下之25 隻小鼠，經剪尾萃取genomic DNA 進行PCR 分析後，確認有1 隻小鼠係攜帶tet-on 表現載體基因與綠螢光蛋白及PDGF-C 之反應載體基因;後者試驗中合計獲得65 隻小鼠出生，其中有7 隻確認係帶有tet-on 表現載體基因及PDGF-C之反應載體基因者。此等轉基因小鼠均已陸續發育成熟，刻正進一步提供針對PDGF-C之生物功能，包括：PDGF-C 表現對於卵巢周期性生理運轉與PDGF-C 表現可能涉及新血管對於胚成功埋殖等之影響，進行詳實深入探之討。Several inducible gene expression systems have been developed in vitro in recent years to overcome limitations with traditional transgenic mice. One of these is the tetracycline-regulated system that has been used successfully in vivo, of which the expression of the targeted protein has been regulated by the promoter sequences containing tetracycline responsive element. To meet these purposes, two transgenes were first constructed, including the former ones carries a highly specific transcription factor (Tet- responsive promoter) and the latter ones carries both of the target gene of PDGF-C regulated by a Tetresponsive promoter and the other target equipped with a double genes containing both the PDGF-C as well asthe EGFP reporter sequences. Based on these, transgenic (Tg) mice harboring the transgene of PDGF-C assigned to be driven by tetracycline were generated after the microinjection strategy using a tetracycline-controlled transactivator (rtTA) and a rtTA-dependent promoter linked to a PDGF-C gene. These allow the expression of PDGF-C be well regulated by the tetracycline and/or its analogue. Consequently, the tightly regulated PDGF-C expression can reveal the true physiological effect of the gene. Results from initial experiments showed that, by double transgenic vectors co-injection technology (Tet-on together with the transgene of pBi-EGFP-PDGF-C and Tet-on together with the transgene of pTRE2hyg-PDGF-C), followed by verification with PCR technology, in the first group, one of the twenty five new born transgenic mouse contains both transgenes of the Tet-on together with the pBi-EGFP-PDGF-C. On the other hand, results from the later studies appeared that seven out of 65 new born mice were transgenic containing both transgenes of tet-on and pTRE2hyg-PDGF-C. All these transgenic mice have reached sexually mature of age and ready providing for further in vivo studies to elucidate the physiological significance of PDGF-C gene expression on events related to animal reproduction as well as the vasculogenesis involved in embryonic implantation