Construction of VR1 siRNA expression vectors and their silencing effects in the DRG neurons of rats / 中国药理学通报

ABSTRACT

Aim To study the function of VR1 in chronic pain, to construct VR1 siRNA expression vectors and to study their silencing effect in the DRG neurons of rats were detected.Methods The hairpin sequences of siRNAs targeting VR1 gene of rat were designed, and two pairs of oligonucleotide sequence were synthesized. The annealed oligonucleotide fragments were cloned into linearized pRNAT-U6.2/Lenti expression vector and identified by PCR and DNA sequencing.Then, they were co-transfected by lipofectamine into 293T cells.The silencing effects of the lentivector-mediated VR1 siRNAs on the expression of VR1 mRNA were determined by RT-PCR after intrathecal injection in rats.Results DNA sequencing showed that the oligonucleotide fragments were correctly cloned into linearized pRNAT-U6.2/Lenti expression vector and the expression of VR1 mRNA in L4-L6 DRG neurons was inhibited significantly by pRNAT-U6.2/Lenti-siVR1 after intrathecal injection in rats.Conclusion The lentivector-mediated siRNAs are successfully constructed and they inhibit the expression of VR1 mRNA in the DRG neurons of rats, which may provide a potential tool for the further study and treatment of chronic pain.