Abstract:

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-L1 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 COX1 gene.

Key words: 3T3-L1 cells; Tet-On inducible gene expression system; Cyclooxygenas gene; Doxycycline