Establishment of glioma cell line with stable RNA interference vector targeting 14-3-3ζ gene

Objective To construct the specific RNA interference vector targeting human 14-3-3ζ gene and to establish a human glioma cell line stably transfected with the vector.Methods According to 14-3-3ζ DNA coding sequence,the specific RNA interference(RNAi) fragments targeting 14-3-3ζ gene were designed and synthesized,which were cloned in to pSilencer3.1-H1neo plasmid vector,and the shRNA eukaryotic expression vector pSilencer-14-3-3ζ targeting 14-3-3ζ gene was constructed.The pSilencer-14-3-3ζ vector and negative pSilencer-14-3-3ζ vector were transfected respectively into U251 cells by LipofectamineTM 2000,and the transfected cells were selected by G418.Anti-G418 clones were isolated and Western blot methods were carried out for the identification of integration of protein levels.Results The specific shRNA eukaryotic expression vector pSilencer-14-3-3ζ targeting 14-3-3ζ gene was constructed successfully,which was identified by restriction endonuclease digestion and sequencing.Cell clones sorted out with G418 selection which had stably integrated RNAi vector targeting 14-3-3ζ gene were obtained.Conclusion Expression of 14-3-3ζ gene can be suppressed markedly by specific RNA interference vector in U251 cells which provides the experimental foundation for the further study on the biological functions and mechanisms of 14-3-3ζ in U251 cells.