四环素诱导调控DEC1表达的Vero细胞系的构建及鉴定

Generation and characterization of Vero cell lines that inducibly regulate DEC1 expression by tetracyclin

为了构建四环素(tetracycline,Tet)诱导的利用Tet-on诱导表达系统调控的Vero-TR母细胞,建立Tet诱导表达或沉默DEC1的Vero细胞系并加以鉴定。试验将表达Tet阻遏蛋白(Tet repressor protein,TetR)的pc DNA6转染至Vero细胞,通过Blasticidin筛选稳定表达TetR的阳性细胞克隆,即Vero-TetR母细胞系。将克隆有DEC1基因(CDS区的pc DNA4真核表达载体或克隆有特异性靶向DEC1基因的shRNA的p Babe-H1真核表达载体,分别转染至Vero-TR细胞,通过Zeocin或Puromycin筛选诱导表达或沉默DEC1的阳性细胞克隆,命名为Vero-DEC1和VerosiDEC1。利用Western blot检测Vero细胞中Tet诱导表达或沉默DEC1的效果。最后成功构建了Vero-TR、Vero-DEC1和Vero-siDEC1的稳定细胞系,Western blot结果显示DEC1能够在Tet的调控下有效表达或沉默。Vero-TR母细胞系的成功构建为建立其他宿主细胞基因表达或沉默的细胞系提供了重要材料,Vero-DEC1和Vero-siDEC1细胞系的成功构建为研究DEC1在正常或感染等应激情况下的生物学功能奠定了基础。

This study aimed to generate tetracycline-induced Vero-TR parent cell line using Tet-on inducible system, and then to generate Vero cell lines that inducibly express or knock down DEC1. pcDNA6 expressing Tet repressor protein （TetR） was transfected into Vero cells, and positive clones that stably express TetR were selected by Blasticidin and designated Vero-TR parental cell line. In addition, pcD- NA4 expressing DEC1 or the pBabe-H1 expressing DEC1 shRNA were transfected into Vero-TR, respectively. The positive cell clones that inducibly express or knockdown DEC1 were screened by Zeocin or Puromycin, and designated Vero-DEC1 or Vero-siDEC1. Moreover, the efficacy of expression or knockdown of DEC1 was detected by Western blot analysis. The stable cell lines of Vero-TR, Veto-DEC1 and Vero -siDEC1 were successfully generated. In addition, the Western blot results showed that DEC1 can be induced by tetracycline to express or knock down. The Vero-TR parental cell line will provide an important material for generating cell lines that inducibly express or knock down any other host cell genes. The Vero-DEC1 and Vero-siDEC1 cell lines obtained in this study lay a critical foundation for studying biological functions of DEC1 under normal or stress conditions of hosts.