Construction of human SH-SY5Y cell line stably transfected with GSK-3β gene silencing expression by lentivirusmediated RNA interference

【Objective】To construct lentiviral vector of RNA interference targeting GSK-3β gene and establish neuroblastoma tumor cell(SH-SY5Y) line model with GSK-3β gene silencing expression.【Methods】According to GSK-3β mRNA, we designed and synthesized the target gene short hairpin RNA(shRNA) sequences and constructed a lentiviral vector. The recombinant lentiviral vectors were confirmed by restriction enzymes, PCR and sequencing. 293T cells were co-transfected with lentiviral vector and packaging system. SH-SY5Y cells were divided into experimental group(infected with the GSK-3β shRNA), Lenti-NC group(infected with control-shRNA) and blank control group(without transfection). Fluorescence microscope was used to observe the transfection efficiency. Real time PCR and Western blot were used to examine the expression of GSK-3β mRNA and protein in SH-SY5Y cells. 【Results】The recombinant lentivirus vector of GSK-3β was successfully constructed and the lentivirus can infect cells more than 90%. Compared with the Lenti-NC group and blank control group, the expression of GSK-3β mRNA and protein was significantly down-regulated in experimental group. 【Conclusions】Human SH-SY5Y cell model with GSK-3β gene silencing expression by lentivirus-mediated RNA interference were effectively established,which could provide a good test cell model for in vitro evaluation for drug or behavioral treatment of AD after GSK-3β gene silence.