Construction of eukaryotic expression recombinant plasmid pEGFP-Mfn2 and transfected into human breast cancer cell line MCF-7

OBJECTIVE: To observe the affect to cell proliferation, we we construct eukaryotic expression plasmid including Mfn2 gene and transfect it inti breast cancer cells MCF-7. METHODS: 1. We constructed the pEGFP-Mfn2 plasmid and transfected it into human breast cancer cell line MCF-7 using liposomes. 2. Using the method of DNA sequencing, RT-PCR, Flow Cytometry and Immunocytochemistry to identify whether the vector transfected. 3. MTT was used to detect how the Mfn2 affect the cell proliferation after transfecting exogenous Mfn2 gene into the cell cycle of MCF-7 cell in vitro. RESULTS: 1. The recombinant plasmid pEGFP-Nl successfully loaded full-length mouse Mfn2 gene encoding, DNA sequencing results were basically consistent with the expected design. General PCR Agarose Gel Electrophoresis of transfected MCF-7 cells were in the presence of exogenous Mfn2 expression. 2. The Agarose Gel Electrophoresis (AGE) showed that the expression of Mfn2 in experimental group was significantly higher than in the control group. After transfection, the cell proliferation velocity and the OD value of pEGFP-Nl group and non-transfection group were basically at equal pace. But the pEGFP-Mfn2 group' 48 h was manifestly less than both of pEGFP-Nl group and non-transfection group(P<0.05). 3. The Immunohistochemical was showed that cancer cells appeared nuclear fragmentation. CONCLUSIONS: The eukaryotic expression plasmid pEGFP-Mfn2 was successfully constructed, and Mfn2 was expressed in MCF-7 cells after being transfected. Exogenous Mfn2 strongly inhibited cell proliferation in MCF-7 cell line.