Construction of shRNA lentiviral expression vector of DFC1 gene and establishment of its stable transfection glioma cell line

WEI Xuehui; LI Xiaoming; ZHANG Yao; RU Yi; WANG Qinhao; LI Xia; LIN Wei

Abstract：Objective The differentiated embryo-chondrocyte expressed gene 1 (DEC1)-short hairpin RNA (shRNA) lentiviral expression vector and stably knock down DEC1 in human gliora cell line T98G were constructed.Methods According to the cDNA sequence of the DEC1 gene provided by the GenBank,two specific shRNA sequences were designed and synthesized.A non-specific sequence was designed as negative control.They were cloned into the Psi-LVRU6MP plasmid vector.After the recombinant vectors were identified by enzyme digestion and DNA sequencing,they were packaged to obtain the lentiviral particles.Then T98G glioma cell line was infected with the three groups of lentivirus.After screening with puromycin,the expression of mCherry was observed under the fluorescence microscope,and the expression level of DEC1 protein in each group was detected by Western Blot.Results shRNA lentiviral expression vector targeting DEC1 gene was confirmed by restriction endonuclease digestion and DNA sequencing.Fluorescence microscopy showed that each group of cells had been efficiently infected by shRNA lentivirus.Western Blot results showed that DEC1 protein levels in the two DEC1-shRNA groups were significantly down-regulated compared with the untreated group and the negative control group,accounting for 39.47％ ±0.69％ and 41.17％ ±0.89％ of the untreated group,respectively (P ＜0.05).But there were no significant differences either between two DEC1-shRNA groups or between the negative control and the untreated group (P ＞0.05).Conclusion The DEC1-shRNA lentiviral expression vector was successfully constructed and stably infected into human glioma T98G cell line.Both groups of DEC1-shRNA lentivirus could markedly suppressed the expression of target gene DEC1.The results provide the experimental basis for further study of DEC1 function and mechanism in the human glioma cell line T98G.