Construction of the eukaryotic expression vector pIRES2-EGFP-p16 and its expression in glioma cells

Objective To construct the eukaryotic expression vector pIRES2-EGFP-p16,and to express p16 in glioma cell C6.Methods The p16 gene was amplified by PCR using pCMV5-HA-p16 as a template,and confirmed by DNA sequencing.The eukaryotic expression vector pIRES2-EGFP-p16 was constructed by introducing p16 DNA fragment into the sites of BamHⅠand XhoⅠof pIRES2-EGFP vector.The plasmid was transfected into the C6 cells using lipofectamine. The expressed EGFP was observed under fluorescent microscope and the P16 protein expression was detected by immunostaining using anti-P16 antibody.Results The eukaryotic expression vector pIRES2-EGFP-p16 was constructed and transfected successfully into C6 glioma cells.The green fluorescence of EGFP was observed in the plasma and nuclei of transfected cells, and P16 protein was found in the plasma and nuclear.Conclusion The recombinant expression vector pIRES2-EGFP-p16 was constructed, and the EGFP and p16 gene could be co-expressed in the C6 cells.This study laid a foundation for the further research of the function of p16 in cell differentiation, growth and tumorigenesis.