Construction Of Lentiviral Vector SiRNA Of APP695 Gene And Its Effect On The APP695 Gene Expression In SH-SY5Y Cells

Aim:To construct the siRNA lentiviral vector of APP695 gene, and detect its effect on APP695 gene in human neuroblastoma SH-SY5Y cell line.The research may present gene therapy of Alzheimer's disease.Methods:Disigned and synthesized siRNA oligonucleotides which targeted APP695 gene.at the same time, the negative control oligonucleotides were systhesized. The synthesized sequences of the oligonucleotides were formed a double-stranded DNA through annealing. The pFU-GW-iRNA plasmid was digested by Hpa I,Xho I,and then the linear plamid was produced. It was the double-stranded DNA that was inserted the linear plamid, and the recombination of APP695-siRNA expression plasmid was generated. The recombinant were transfected into the susceptible cell E.coli DH5α,and the positive colony were choosen. Then, the positive clony were confirmed by PCR and DNA sequencing. Meanwhile, the negative control recombinant was constructed.392T cells were cultured and cotransfected with lentiviral vector pFU-GW-iRNA and packing plasmids,to produce lentiviral vector which can infect other cells. The SH-SY5Y cells were divided into APP695-siRNA group,control group and negative control group. The APP695mRNA was detected by fluorescence quantitative Real-time polymerase chain reaction (FQ-RT PCR).Results:①The positive clones of recombinants were identified by PCR and DNA sequencing,the result showed that the target oligonucleotide was accuratly cloned into pFU-GW-iRNA plasmid.②The plasmids intransfected the 293T cells and lentiviral particles were harvested.③The SH-SY5Y cells were transfected by lentivius vectors afer 72h, the APP695mRNA expression of APP695-siRNA group was obviously lower than the control group and negative control group (p<0.001) there was statistical significance. But the different between the control group and negative control group was no significant (p=0.112).Conclusion:The siRNA sequence target of APP695 gene was accurately cloned into the pFU-GW-iRNA plasmid and APP695-siRNA lentivirus vector was constructed successfully.The APP695-siRNA lentiviral vector effectively silenced the APP695mRNA expression in SH-SY5Y cells.