Construction of a lentiviral vector of RNA interference of PERK gene and identification in human dental pulp cells

doi:10.19439/j.sjos.2017.04.003

Abstract

Abstract: To construct an expression vector of a small hairpin RNA (shRNA) targeting human PERK gene and to observe gene-silencing effects of PERK in human dental pulp cells (DPCs). METHODS: According to PERK gene cDNA sequence, shRNA was designed and synthesized, which was then annealed into hU6-MCS-CMV-EGFP vector. After identified by sequencing, hU6-MCS-CMV-EGFP vector and packaging vector were co-transfected into 293 T cells. 72 hours later, the recombinant lentiviruses were obtained after harvesting and concentrating. Then LV-PERK-RNAi vectors were transfected into DPCs at an appropriate multiplicity of infection. To verify the interference effect, real- time PCR and Western blot were used to detect expression levels of PERK mRNA and protein in the transfected DPCs. The data were analyzed with SPSS 24.0 software package. RESULTS: LV-PERK-RNAi vectors were successfully constructed with a high titer of 3×108 TU/mL. The results of RT-PCR and Western blot demonstrated that after infection with LV-PERK-RNAi vector at a multiplicity of infection of 30, the expression level of PERK gene in DPCs was significantly down-regulated compared with control group. At mRNA level, the interference rate was about 63%. CONCLUSIONS: An effective lentiviral shRNA expression vector targeting the PERK gene is successfully constructed and can be used for further study on the function of PERK gene.

Key words: PERK, RNA interference, Lentiviral vector

CLC Number:

R781.31

WEN Yang, ZHU Ya-qin. Construction of a lentiviral vector of RNA interference of PERK gene and identification in human dental pulp cells[J]. Shanghai Journal of Stomatology, 2017, 26(4): 363-367.

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