tTS/rtTA系统下调目的基因基础表达的细胞模型研究

Reducing Background Expression of Target Gene with tTS/rtTA System in Cell Model

【目的】构建带有肝脏特异性启动子和tTS沉默子的四环素调控系统,观察其在人HepG2细胞内的表达,建立严密型四环素调控系统的HepG2细胞模型。【方法】通过二次分子克隆构建重组真核表达质粒载体pliv7-rtTA-tTS-Neo,用脂质体法将质粒pliv7-rtTA-tTS-Neo及pliv7-rtTA-Neo转染人HepG2细胞,经G418筛选后的克隆用有限稀释法进行单克隆化,克隆扩增后瞬时转染pTRE-Luc质粒,强力霉素(1mg/L)诱导表达后检测荧光素酶活性,挑选出低背景和高诱导表达的HepG2细胞克隆。【结果】带有tTS的细胞株其诱导倍数明显高于不带有tTS的细胞株(192︰21),其中克隆9的诱导倍数最高(256),获得1株高表达、低背景的HepG2/pLiv7-rtTA-tTS-Neo细胞株。【结论】荧光素酶活性检测结果验证了tTS对四环素调控系统的内在泄露问题有一定的抑制作用,使四环素调控系统的开关具有真正意义上的严密性。

[ Objective ] To construct a tetracycline-controlled expression system with liver-specific promoter and tTS silencer and investigate its expression in the human HepG2 cells; to establish a cell model with tight tetracycline-controlled expression system. [Methods] Recombined eukaryotic plasmic vector, pliv7-rtTA-tTS-Neo, was constructed by subcloning and transfected into HepG2 cells. 14 days following transfection, G418-resistant clones were attained, monocloned and transiently transfected with pTRE-Luc by using lipofectamine. Luciferase reporter assay of HepG2/ pLiv7-rtTA-tTS-Neo and HepG2/pLiv7-rtTA- Neo were detected in the presence of doxycycline（ 1 mg/L） to select one clone with low background and high induction. [Results] The HepG2 cell line with tTS was more inducible than that without tTS （192 ： 21 ）. Clone 9, which exhibited high level of induction and high gene expression level, was obtained. [ Conclusion ] The result of luciferase reporter assay confirms that tTS can restrain the leaky in the tetracycline-controlled expression system. This expression system made the on-off of tetracycline-controlled expression system more efficient.