Establishment of glioma cell line with stable RNA interference vector targeting MSP58 gene

Objective To explore the establishment of glioma cell line with stable RNA interference vector targeting MSP58 gene.Method According to MSP58 cDNA coding sequence, the specific RNA interference (RNAi) fragments targeting MSP58 gene were designed and synthesized, which were cloned into pSilencer3.1-H1neo plasmid vector, and the shRNA eukaryotic expression vector pSilencer-MSP58 targeting MSP58 gene was constructed. The pSilencer-MSP58 vector and negative pSilencer-MSP58 vector were transfected respectively into U251 cells by LipofectamineTM2000, and the transfected cells were selected by G418. Anti-G418 clones were isolated and Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot methods were carried out for the identification of integration of RNAi vectors in mRNA and protein levels.Results The specific shRNA eukaryotic expression vector pSilencer-MSP58 targeting MSP58 gene was constructed successfully, which was identified by restriction endonuclease digestion and sequencing. Six cell clones sorted out with G418 selection which had stably integrated RNAi vector targeting MSP58 gene were obtained. Conclusion MSP58 gene expression can be suppressed markedly by specific shRNA in U251 cells.