Design of Short Interference RNA That Target the Pain Gene Pkc Gamma

RNA interference is a powerful genetic approach for efficiently silencing target genes. One advantage of this technique is the high specificity it offers over pharmacological antagonism in that it cleaves the mRNA of a precise protein. The purpose of this study is to use RNA interference to target PKC Gamma, a protein kinase that has been identified in neurons of the brain and spinal cord and implicated in the production of persistent pain. Firstly, we designed five 21 base pair small interfering RNAs (siRNA) that target the gene that encodes the mouse PKC Gamma by using the MIT-Whitehead siRNA design program. Next, the siRNAs were subjected to a NCBI BLAST search that checks the specificity of each siRNA for the targeted gene and identifies similar sequences in other genes. To screen for effective siRNAs, a small hairpin RNA (shRNA) will be constructed from potential siRNAs by the addition of a stem loop structure. The shRNA is then cloned into the pAAV plasmid with a H1 promoter. Subsequently the PKC Gamma cDNA (mouse) is inserted into the psiCHECK vector, containing the reporter genes renilla luciferase (Rluc) and firefly luciferase. Both plasmids will be co-transfected into HEK 293 cells. Effective shRNAs will cleave the PKC Gamma cDNA, which will decrease the luminescence of Rluc in comparison to the firefly. Effective shRNA will be incorporated into viral vectors to be injected into animals. A successful PKC Gamma siRNA will prevent or reduce pain in a mouse model.