Construction of Eukaryotic Expression Vector Containing Mouse Fractalkine Gene and Its Expression in Mouse Hepatocellular Carcinoma Cell Line

Objective: To clone mouse fractalkine cDNA, construct its eukaryotic expression vector and to express it in mouse hepatocellular carcinoma cell line. Methods: The total RNA was extracted from mouse breast cancer cell line D2F2. The full-length cDNA encoding mouse FK gene was obtained by RT-PCR method and inserted into pCR2. 1 TOPO cloning vector. After the sequencing was confirmed, the gene was subcloned to pIRES to construct recombinant eukaryotic expression vector pIRES-FK. The recombinant plasmid was transfected into MM45T. Li cells by lipofectamine method and positive cell clones were screened with G418. Expression of mouse FK gene in the transfected cells was confirmed with RT-PCR and immunocytochemistry. Results: Enzyme digestion analysis and sequencing showed that the target gene was cloned into recombinant vector. Expression of mouse FK gene in the transfected MM45T.Li cells was identified with RT-PCR and immunocytochemistry. Conclusion: The eukaryotic expression plasmid containing mouse FK gene is successfully constructed. The positive MM45T.Li cell clones expressing mouse FK gene are obtained, which may be a promising cell model for studying.