Establishment of CHO Cell Strain for Overexpression of bcl-2 Gene

Objective To establish a CHO cell strain for overexpression of bcl-2 gene.Methods Total RNA was extracted from CHO cells and used as a template for amplification of bcl-2 gene by RT-PCR.The amplified bcl-2 gene was cloned into vector pcDNA3.1,and the constructed recombinant plasmid pcDNA3.1-bcl-2 was transfected to CHO-dhfr-cells,and positive clones,named as CHO-G6 cells,were screened with neomycine,then determined for bcl-2 gene expression and apoptosis by flow cytometry,and the proliferation activity by MTT method.Results The percentage of CHO-G6 cells positive for bcl-2 gene expression(92.46%)was significantly higher than that of CHO-dhfr-cells(76.08%),while the apoptosis rate of the former(1%)was significantly lower than that of the latter(19%).However,the proliferation activities of CHO-G6 cells after culture in serum-free medium for 1 ～ 5 d were significantly higher than those of CHO-dhfr-cells.Conclusion A CHO cell strain for overexpression of bcl-2 gene,with a certain anti-apoptotic activity,was successfully constructed,which laid a foundation of production of recombinant protein.