Construction Of Shrna-expressing Vectors Targeting To Rat Delta Opioid Receptor And Analysis Of Its Effects

Objective : To design and construct short hairpin RNA (shRNA) eukaryotic expression plasmids targeting DOR gene which may play an important role in morphine tolerance, and to select the plasmids that silence DOR gene most efficiently.Methods:Three pairs of short chain oligonucleotides targeted to rat DOR mRNA (Accession No: NM₀12617) were designed and synthesized individually based on the sequence of rat DOR mRNA at first. The synthestic sense and antisense oligonucleotide strands were mixed together in annealing buffer to form 3 DNA duplexes. Then the DNA segments were cloned into pGenesil-1.2 vectors respectively and the plasmids were identified by restriction analysis and sequencing test. Subsequently the plasmids were transfected into pheochromocytoma cell (PC12 celll) using lipofectamine2000 reagent, and the transfection rate of recombinant plasmids was measured 48 h after transfection. At last the mRNA and protein expressions of DOR were analyzed by RT-PCR and Western Blot technique, and compared with that of nontransfection group and negative control plasmid.Result: The results of restriction analysis and sequencing test showed that all three designed DOR shRNA-expression duplexes were successfully inserted into the plasmid vector pGenesil-1.2 respectively and recombinant plasmid vectors were constructed meeting our aims. The transfection rate of recombinant plasmids in PC12 cells was approximately 60%. Forty-eight hours after transfection, the DOR-mRNA and protein levels of shRNA3 group were significantly lower than that of the blank control and that of the negative control group.Conclusion:The DOR-shRNA expression vectors were constructed and selected successfully and could be used in RNAi's study on morphine tolerance.