受四环素调控的真核表达系统SK-Hep1细胞株的建立

建立稳定、高效表达外源基因的SK-Hep1细胞株,以便进一步研究基因的作用.首先将调控质粒pCDNA6/TR转染SK-Hep1细胞,经潮霉素筛选得到多个稳定单克隆.各个单克隆分别扩大培养后,转染pCDNA4/TO/lacZ质粒,再经过DOX(强力霉素)诱导表达,检测β-半乳糖苷酶(β-D galaetosidase,β-gal)活性,从而筛选出高诱导水平低背景表达的SK-Hep1 tet-on细胞株.最后,再将pCDNA4/TO/c-myc质粒转染进SK-Hep1 tet-on细胞株,进一步通过Western blotting检测该系统对下游基因的表达调控.成功建立了一株受DOX调控的高诱导水平低背景表达的细胞株SK-Hep1 tet-on 10#.

Establishment of a Tetracycline-inducible System in Human Hepatoma Cell Line SK-Hep1

It was to establish a liver cancer cell line,SK-Hep1 tet-on with gene expression regulated by DOX,which is useful tool for investigations of gene function and gene transfer.SK-Hep1 cells were transfected with pCDNA6/TR 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 Dox after all clones being transfected with pCDNA4/TO/lacZ for clones.Finally,SK-Hep1 tet-on cells were transfected with pCDNA4/TO/c-myc using Liposome2000 transfection reagent,and the transfected cells were treated with DOX for 24 h.The expression level of c-myc was detected by Western blotting.SK-Hep1 tet-on cell line which exhibits low background and high induction of luciferase in response to DOX was established.