- 期刊
Establishment of a DNA vector system for doxycycline-inducible and β-cell-specific transgene expression
建立以胰島素基因啟動子啟動可誘導式基因表現之DNA載體系統
摘要
A single vector containing a complete tetracycline-on (Tet-on) inducible system was proposed and constructed with both regulatory and responsive elements. The developed Tet-on inducible vectors contain a Tet-sensitive transactivator gene (rtTA) under the control of the rat insulin II gene promoter (RIP) or porcine insulin gene promoter (PIP), a transgene cloning site, which is under the control of Tet operator sequence (tetO) flanked with a minimal CMV promoter (TetCMVmin). A neomycin resistant gene expression cassette was also included for screening stable clones in the future. With the developed Tet-on inducible vector, the green fluorescent protein (GFP) as a reporter gene could be effectively induced in HIT-T15 cells, a hamster pancreatic β-cell line, by the treatment of doxycycline (Dox), and is also responsive to glucose in the regulation of transgene expression, demonstrating the versatility of this single vector-based inducible system in β-cell specific expression of transgene, whose transactivator is controlled by RIP or PIP. In contrast, it could not lead to the expression of GFP in DK cell (duck kidney cell line) in the presence of Dox. In conclusion, the developed Tet-on inducible vector exhibits a high potential in controlling transient gene expression and the generation of transgenic animal models.
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。本試驗構築一簡便DNA載體系統，具有可誘發性基因表現及β-細胞特異性表現，主要DNA原件含有可受大鼠及豬胰島素基因啟動子調控之四環黴素調節性DNA片段，以及可表現外源基因如綠螢光蛋白的四環黴素反應性DNA片段。將此DNA載體系統轉殖入大鼠胰臟β-細胞株後，添加四環黴素確實可有效誘發外源基因綠螢光蛋白的表現，也受培養液葡萄糖濃度所調控其表現，但此外源基因綠螢光蛋白卻不能被誘發表現於鴨腎臟細胞株。這些結果證明我們已經建構可應用於β-細胞特異性表現之可調節式基因專一表現系統。