[Establishment of a transgenic cell line with stable expression of human CD14].

ABSTRACT

OBJECTIVE: To establish a transgenic cell line with stable expression of CD14. METHODS: Total RNA extracted from peripheral blood mononuclear cells was treated with RNAase-free DNAase, the human CD14 gene was cloned and sequenced through the RT-PCR, T-A clone techniques and ABI PRISM377 machine. Eukaryotic expression vector pcDNA3.1(+)/CD14 was constructed by cleaving with double restriction endonucleases EcoR I/Xba I and ligating with T4 ligase. The human cervical cancer cell line Hela was transfected with the positive recombinant plasmid pcDNA3.1(+)/CD14 using superfect transfection reagent. Positive clones were selected by G418 at a concentration of 0.5 µg/µl and the expression of human CD14 on the transfected Hela cells was confirmed by quantitative PCR and immunofluorescent assay. RESULTS: There was significantly difference om expression of CD14 mRNA between the blank pcDNA3.1(+) transfected cells and pcDNA3.1(+)/CD14 transfected cells (P<0.01). The fluorescence was significantly stronger on the stable cell line Hela-CD14 than that on the transiently transfected Hela cells,and no visible fluorescence was observed in blank vector transfected cells. CONCLUSION: The transfectant cell line Hela-CD14 with stable expression of human CD14 has been successfully established, which can be used to study human CD14 molecular and CD14-associated monocyte/macrophage cell diseases.