- 期刊
A DNA vector system for MMTV-promoter-based, doxycycline-inducible transgene expression and verified by expressing Mst4 gene
建立以小鼠乳腺腫瘤病毒基因啟動子啟動可誘導式基因表現DNA載體系統並表現Mst4基因
摘要
A simplified tetracycline-on (Tet-on) system including all the required regulating elements was constructed in a single vector (poMRT_(neo)/GFP plasmid). The single vector contains a Tet-controlled transactivator gene, rtTA2s-M2, a glucocorticoid-responsive mouse mammary tumor virus (MMTV) promoter, a green fluorescent protein (GFP) reporter gene under the control of Tet operator sequences (tetO) flanked with a minimal CMV promoter (TetCMVmin) and a mammalian selection marker neomycin resistant gene. When tested in NMuMG, a mouse mammary epithelial cell line, the expression of GFP was low without treatment and could be efficiently elevated with treatment of doxycycline (Dox, a Tet analogue). Importantly, MMTV-promoter-based Tet-on inducible vectors enabled the transgene not only to express in a mouse mammary cell line, but also be tightly regulated under the simultaneous treatment with Dox and dexamethasone (Dex, a glucocorticoid analogue), when culture media without steroid. Next, in order to verify the construction strategy is correct, we replaced GFP with Mst4 gene, a sterile 20-like kinase, including a full length wild-type cDNA form and two c-terminus deletion mutants in this inducible system (plasmids poMRT_(neo)/HA-Mst4, poMRT_(neo)/HA-Mst4△272, poMRT_(neo)/HA-Mst4△297, poMRT_(neo)/ GFP-Mst4, poMRT_(neo)/GFP-Mst4 △272 and poMRT_(neo)/GFP-Mst4 △297). The resulting vector could display the Mst4 expression with lower basal level, higher inducibility and its nucleocytoplasmic dissection. These data demonstrated that we have created a MMTV-promoter-based, doxycycline-inducible transgene system and the simple system is suitable for expressing foreign gene in mammary cells.
並列摘要
四環黴素可誘導系統（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。本研究利用已構築只需一個DNA質體就可具有四環黴素可調節式基因表現（poMRT_(neo)/GFP plasmid），當轉殖入乳腺上皮細胞株後發現須同時添加四環黴素及醣質皮質素方能誘發表現，證明醣質皮質素確實會作用於MMTV啟動子。之後將此質體應用到表現功能性基因上，以Mst4蛋白質基酶基因及其c端刪除突變序列取代原有GFP報導基因， 得到poMRT_(neo)/HA-Mst4，poMRT_(neo)/HA-Mst4△272，poMRT_(neo)/HA-Mst4△297，poMRT_(neo)/GFP-Mst4，poMRT_(neo)/GFP-Mst4△272 and poMRT_(neo)/GFP-Mst4△297等質體DNA。當添加此四環黴素後展現高度誘發Mst4表現，並且所表現的Mst4蛋白質激酶能正常分佈細胞胞器內。這些結果證明我們已經建構可應用於乳腺上皮細胞之可調節式基因專一表現系統。