Both humanized mouse lines were obtained as previously described25. Briefly, the line homozygous for the 118A allele (h/mOPRM-118AA, wild-type) was generated by replacing exon 1 of the mouse MOR gene with the corresponding human sequence. The line homozygous for the human 118G allele (h/mOPRM-118GG, mutant) was generated by employing site-directed 118A→G mutagenesis on the same construct, followed by replacement of exon 1. Except for the A-G exchange, therefore, both lines are genetically identical. The humanized mouse lines were generated on C57/BL6 background and the colony is maintained by h/m OPRM1-118AG breeding in order to generate littermates of all three genotypes: h/m-OPRM1-118AA, h/m-OPRM1-118AG, and h/m-OPRM1-118GG. The allele-frequencies in the offspring followed Mendelian distribution (24.1% h/m-OPRM1-118AA, 25.3% h/m-OPRM1-118GG, and 50.6% h/m-OPRM1-118AG) and were equally distributed with regards to sex of offspring (52.2% female, 47.8% male). Currently, the line is in the 7th generation of breeding and no adverse phenotype has arisen during this time. Heterozygous mice were not studied so that both extremes could be compared and allow us to maintain a focused interpretation of the data.
