受强力霉素调控的真核表达系统RAW264.7 Tet-lon细胞株的建立  

Establishment of a RAW264.7 Tet-on cell line and its gene expression regulated by doxcycline

作　　者：彭晓慧[1] 毛萱[1] 汤顺清[1] 

机构地区：[1]暨南大学生命科学技术学院生物医学工程系,广东广州510632

出　　处：《暨南大学学报（自然科学与医学版）》2011年第4期383-386,共4页Journal of Jinan University(Natural Science & Medicine Edition)

基　　金：国家863项目(2007AA09Z436)

摘　　要：目的:利用Tet-lon诱导表达调控系统构建可以用强力霉素调控表达的RAW264.7 Tet-lon细胞株,探讨目的基因NF-kB的作用。方法:将调控质粒pCDNA6/TR转染RAW264.7细胞,经潮霉素筛选得到稳定单克隆。单克隆扩大培养后,转染pCDNA4/TO/lacZ质粒,再经过强力霉素诱导表达,检测β-半乳糖苷酶(β-gal)活性,筛选出受强力霉素调控的高诱导水平、低背景表达的RAW264.7 Tet-on细胞株。结果:成功建立一株受强力霉素调控的高诱导水平、低背景表达的细胞株RAW264.7 Tet-on 3,并且强力霉素对下游基因β-gal的诱导效果具有浓度和时间依赖性,强力霉素的最佳诱导时间为24 h,强力霉素的最佳诱导质量浓度为1μg/mL。结论:已获得受强力霉素调控的细胞株。Aim：To establish a macrophage cell line,RAW264.7 Tet-on with gene expression regulated by doxcycline,which may be used tool for investigations of gene function and gene transfer.Methods： RAW264.7 cells were transfected with pCDNA6/TR by using liposome2000 transfection reagent,and the transfected cells were selected with hygromycin.The hygromycin-resistant clones were isolated and cultivated.The clones were selected with high induction of luciferase and low background in response to doxcycline after all clones were transfected with pCDNA4/TO/lacZ.Results： RAW264.7 Tet-on cell line which exhibits low background and high induction of luciferase in response to doxcycline was established.doxcycline induced the expression of downstream genes in a concentration-dependent and time-dependent manners.The best induction period is 24 hours,and the best induction concentration is 1 ug/mL.Conclusion：We have successfully established a cell line which can be regulated be doxcycline,and these results laid the foundation for further study on the biological functios of certain genes,including NF-kB and so on.

关 键 词：强力霉素 转染 诱导表达调控 RAW264.7细胞株 

分 类 号：R978.14[医药卫生—药品]