Construction of shRNA lentivirus vector targeting rat PKCγ gene and identification of its interference efficiency

0bjective: Protein kinase C(PKC),especially PKCγ,is reportedly involved in the nociceptive processing at the spinal level.This study was designed to construct the shRNA lentivirus vectors of the rat PKCγ gene and identify their interfering efficiency at the cell level.Methods: Three target sequences(19nt) were selected according to the PKCγ-mRNA sequence and the sense and antisense oligo-nucleotides were designed and synthesized.After annealing,these double DNA strands were cloned to the pLVTHM vector that contained the H1 promoter and green fluorescent protein(GFP).Meanwhile,an unrelated sequence was used as the negative control.All virus stocks were produced by transfection of 293T cells with the vector plasmid,pMDLg-pRRE,Rsv-REV and pMD2G.Then the lentivirus was transduced to the C6 cells and the inhibition efficiency of the recombinant shRNA lentivirus vector was identified by Western blotting.Results: The results of DNA sequencing confirmed the successful construction of the 3 shRNA lentivirus vectors,among which pLV-PKC2(site: +1913-+1931) possessed the highest inhibition efficiency.Conclusion: The shRNA lentivirus vectors of pLV-PKC1-3 were constructed successfully and the shRNA lentivirus vector of pLV-PKC2 could specifically and highly efficiently down-regulate the PKCγ gene expression.