Molecular NeuroscienceResearch PaperComplete knockout of the nociceptin/orphanin FQ receptor in the rat does not induce compensatory changes in μ, δ and κ opioid receptors
Section snippets
Animals
All experiments were approved by the Animal Care Committee of the Royal Dutch Academy of Sciences and conducted according to the Dutch law for animal experiments. All efforts were made to minimize the amount of animals. NOPr knockout rat (OPRL11Hubr) was generated by target-selected ENU-induced mutagenesis in a Brown Norway background (for detailed description, see Smits et al., 2006). The animals were outcrossed for two generations on a Brown Norway background. Because Brown Norway rats breed
NOPr is completely absent in oprl1−/− rats
High-throughput resequencing of target genes in a library of ENU-mutagenized Brown Norway rats (Smits et al., 2006) revealed a C to G transversion at position 3657 in the oprl1 gene (ENSRNOG00000016768), resulting into a premature stop codon (TAC>TAG) in the third exon. Analysis of [3H]N/OFQ binding revealed that NOPr expression is completely absent in rats homozygous for the oprl1 gene premature stop codon (Fig. 2), indicating that this premature stop codon results into a full knockout of
Discussion
Using recently established gene knockout technology (Smits et al 2006, Smits et al 2008, van Boxtel et al 2008b) we obtained a rat knockout for NOPr. We show that the ENU-induced premature stop codon results into a complete lack of NOPr in homozygous knockout rats. Importantly, no changes were found in μ, δ and κ-opioid receptor binding, arguing that this rat model provides selectiveness towards the inactivation of NOPr and that oprl1−/− rat knockout phenotypes are not likely to be confounded
Conclusion
We conclude that the premature stop codon in the rat oprl1 gene results into a complete knockout of NOPr. The absence of compensatory adaptations in μ, δ and κ opioid receptor expression makes this animal model valuable in the further understanding of NOPr function, its interaction with other neurotransmitter systems, and the assessment of the therapeutic effects of N/OFQ (ant)agonists in pain, Parkinson's disease, and drug addiction (for extensive review see for exampleCiccocioppo et al 2000,
Acknowledgments
We would like to acknowledge Pim Toonen and Mark Verheul for genotyping of animals. This work is funded by the Netherlands Organisation for Scientific Research (NWO), grant # 91676160, awarded to J. R. Homberg and the award “Exploiting natural and induced genetic variation in the laboratory rat” to E.C. from the European Heads of Research Councils and European Science Foundation EURYI (European Young Investigator) Award scheme. NWO, the European Heads of Research Councils, and the European
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2015, European Journal of PharmacologyCitation Excerpt :Male Sprague–Dawley and Wistar rats were supplied by Harlan (Varese, Italy). Male NOP(−/−) rats supplied by GenOway (Lyon, France) were generated in a Brown Norway background and subsequently backcrossed on a Wistar background for eight generations as previously described in detail (Homberg et al., 2009). For knockout studies Wistar rats were used as controls and indicated as NOP(+/+).