Tet-on系统诱导COX1基因在小鼠前脂肪细胞3T3-L1中的表达

Conditional expression of COX1 gene in 3T3-L1 predipocytes of mice by Tet-On system

目的利用Tet-On诱导表达系统,调控目的基因环氧化酶(COX)的表达,探讨COX对脂肪细胞分化的作用,为进一步深入研究肥胖的致病机制奠定理论基础。方法脂质体介导调控质粒pTet-On转染3T3-L1细胞,G418筛选单细胞克隆,RT-PCR法检测四环素反应转录活化因子(rtTA)基因的整合;pTRE-Tight-Luc质粒瞬时转染含rtTA基因的细胞克隆,强力霉素(DOX)诱导表达后检测荧光素酶活性,筛选出高效表达的抗性克隆,命名为3T3-L1-Tet-On-26#;构建重组质粒pTRE-Tight-COX1,双酶切鉴定后测序,将测序正确的重组质粒瞬时转染3T3-L1-Tet-On-26#细胞克隆,免疫荧光检测COX1的表达。结果建立了3T3-L1-Tet-On细胞模型;成功构建了pTRE-Tight-COX1重组质粒,瞬时转染3T3-L1-Tet-On-26#,DOX诱导后COX1表达显著升高。结论诱导表达细胞株3T3-L1-Tet-On的建立为COX1功能基因的研究提供了平台。

Objective To examine the role of cyclooxygenas （COX） gene in adipocyte differentiation regulated by Tet- On inducible gene expression system and further study the pathogenesis of obesity. Methods Liposome was used to mediate transfection of plasmid pTet-on in 3T3-L1 cells, and stable 3T3-Ll cell line expressing tetracycline responsive transcriptional activator（rtTA） gene was selected through G418 screening and RT-PCR method. Plasmid pTRE-Tight- Luc was transiently transfected into 3T3-L1 cells which expressed the rtTA gene. Luciferase activity was detected after doxycycline （DOX） induction, and the positive clone efficiently expressing rtTA gene was named as 3T3-L1-Tet-On-26#. Plasmid pTRE-Tight-COX1 was constructed and examined by double enzyme digestion assay and gene sequencing, and then transiently transfected into 3T3-L1-Tet-On-26# cells. Immunofluorescence method was used to examine the expression of COX1. Results 3T3-L1 stable cell line expressing rtTA gene was established. Recombinant plasmid pTRE- Tight-COX1 was constructed successfully and transfected into 3T3-L1 stable cell line. The expression of COX1 in 3T3- L1 stable cell line was significantly increased after DOX induction. Conclusion The induced expression of 3T3-L1- Tet-On cell line has been established, which will provide a research platform for the COXI gene.