Construction Of Eukaryotic Express Vector With Mouse PPARγ Gene And Its Effect On Breast Cancer MDA-MB-231 Cells Cycle

Objective To obtain eukaryotic express vector of Mouse PPARy gene and detect its expression, subcellular localization and Effect on breast cancer MDA-MB-231 Cells cycle.Methods PPARy cDNA was amplified by RT-PCR from Mouse Liver tissues, Fresh PCR products were cloned into pGEM-T vector and sequenced. Then the recombinant ORF was subcloned into pEGFP-Cl vector and sequenced. The recombinant vector was transfected into MDA-MB-231 cells by lipofectamine2000, the expression level of PPARy mRNA and protein were identified by real-time PCR and western blot, respectively. Immunofluorescence microscope observed the subcellular localization. The flow cytometry analysis the Effect on breast cancer MDA-MB-231 Cells cycle.Results PPARy gene was identified to be inserted into pEGFP-C1 vector correctly and the recombinant vector expressed PPARy mRNA and protein stably. Enhanced Green Fluorescence Protein was expressed in the whole cytoplasm of MDA-MB-231 cells, while, the green fluorescence protein of recombinant eukaryotic expressive vector pEGFP-Cl-PPARy was main expressed in the nucleus and dispersed into the whole cytoplasm in MDA-MB-231 cells. The flow cytometry analysis showed a significant increase of S cell population after transfected with pEGFP-C1-PPARy.Conclusion The eukaryotic expressive vector containing Mouse PPARy gene was successfully constructed and expressed. The subcellular localization of PPARy was main expressed in the nucleus of MDA-MB-231 cells. Exogenous PPARy gene overexpression could block MDA-MB-231 cell cycly in S phase.