Tet-On基因表达系统定量调节荧光素酶基因在CHO细胞中的表达  

Tet On GENE EXPRESSION SYSTEM ACHIEVES QUANTITATIVE CONTROL OF LUCIFERASE GENE EXPRESSION IN CHO CELLS

作　　者：伏爽[1,2] 程康[1,2] 王申五 马大龙[1,2] 王德炳[1,2] 

机构地区：[1]北京医科大学人民医院血液病研究所 [2]北京医科大学分子免疫学研究中心

出　　处：《北京医科大学学报》1998年第6期512-514,554,共4页Journal of Peking University(Health Sciences)

基　　金："九五"国家科技攻关项目

摘　　要：目的：应用ＴｅｔＯｎ基因表达系统建立ＣＨＯＴｅｔＯｎ细胞株，通过强力霉素（Ｄｏｘ）调节荧光素酶基因的表达。方法：ｐＴｅｔＯｎ质粒转染ＣＨＯ细胞株，经Ｇ４１８筛选，得到稳定表达株ＣＨＯＴｅｔＯｎ。ｐＴＲＥＬｕｃ质粒瞬时转染ＣＨＯＴｅｔＯｎ克隆１至３０，培养基中加入２ｍｇ·Ｌ－１Ｄｏｘ或不加Ｄｏｘ，培养７２ｈ后检测荧光素酶活性。结果：克隆１８、２８、２９当培养基中加入Ｄｏｘ（即“Ｏｎ”状态）时荧光素酶活性高，当培养基中不加Ｄｏｘ（即“Ｏｆ”状态）时荧光素酶活性低，故选择克隆１８、２８、２９作为高表达、低背景的ＣＨＯＴｅｔＯｎ细胞株。结论：ＣＨＯＴｅｔＯｎ细胞株的建立，可利用四环素及其衍生物调节多种外源基因的表达，有效调控基因表达的时间和水平，定量诱导毒性蛋白的表达，增加治疗的疗效和安全性，有望为基因治疗提供一条可控的安全途径。Objective: To establish CHO Tet On cell line using Tet On gene expression system, and control luciferase gene expression by doxycycline(Dox). Methods: pTet On was stably transfected into CHO cells. Cells resistant to G418 were cloned, and named as CHO Tet On. pTRE Luc was transiently transfected into CHO Tet On clone 1-30. All clones were grown for 72 hours in the absence or in 2 mg·L -1 of Dox. Different clones achieved different levels of luciferase activity. Results: Clones 18、28、29 had high maximal luciferase expression in 2 mg·L -1 Dox (“On” state), and low basal luciferase expression in the absence of Dox (“Off” state). Clones 18、28、29 were selected as high expression and low background CHO Tet On clones. Conclusion: The ability to modulate the expression of multiple genes by tetracycline and its derivatives is likely to regulate the timing and level of gene expression, quantitatively induce the expression of toxic protein, increase therapeutic efficacy and safety, and provide a safe and regulatable way for gene therapy.

关 键 词：Tet-On基因 基因表达调控 荧光素酶基因 CHO细胞 

分 类 号：Q343.11[生物学—遗传学]