Chinese Journal of Tissue Engineering Research ›› 2011, Vol. 15 ›› Issue (46): 8668-8670.doi: 10.3969/j.issn.1673-8225.2011.46.028

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Construction and identification of delta opioid receptor-short hairpin RNA eukaryotic expression vector

Hu Song-quan, Wang Peng, Yang Hui   

  1. Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
  • Received:2011-04-28 Revised:2011-05-17 Online:2011-11-12 Published:2011-11-12
  • Contact: Yang Hui, Doctor, Associate professor, Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China yanghui@tjh.tjmu.edu.cn
  • About author:Hu Song-quan★, Master, Attending physician, Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
  • Supported by:

    the National Natural Science Foundation of China,No. 30872440*

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Abstract:

BACKGROUND: Long-term application of opioid receptor for treatment of pains would cause drug tolerance or addiction, which is possibly related to upregulated delta opioid receptor (DOR) density.
OBJECTIVE: To design and construct short hairpin RNA (shRNA) eukaryotic expression plasmids targeting DOR gene which may play an important role in morphine tolerance.  
METHODS: Three pairs of short chain oligonucleotides targeted to rat DOR mRNA (Accession No: NM_012617) were designed and synthesized individually based on the sequence of rat DOR mRNA at first. Then the synthestic sense and antisense oligonucleotide strands were mixed together in annealing buffer to form 3 DNA duplexes. Subsequently, the DNA segments were cloned into pGenesil-1.2 vectors respectively. At last, the plasmids were identified by restriction analysis and sequencing test.
RESULTS AND CONCLUSION: 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 DOR-shRNA expression vectors were constructed successfully and could be used in RNAi’s study on morphine tolerance.

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