Figure 1. Generation of mu opioid receptor conditional knockout mice.

(A) The conditional Oprm1 (Oprm1 ^fl) allele was created by homologous recombination. The scheme shows the wild-type Oprm1 allele, the targeting vector, targeted allele and conditional allele obtained after excision of Hygro^r by a Cre recombinase treatment of ES cells. The Oprm1 ^fl conditional allele - or “floxed” allele - harbors two loxP sites flanking the Oprm1 exon 2 and 3. Black boxes, exons; Mf, Mfe1; Sa, SalI, Sp, Spe1 restriction sites; white triangles, loxP sites; Hygro box, floxed hygromycin-resistance cassette, grey box, probe for Southern blot analysis. Dash lines indicate expected labeled DNA fragments in Southern blot analysis. (B) Southern blot analysis of wild-type and targeted alleles in ES cells. Genomic DNA was digested using Mfe1 and hybridized to a 3’ external probe, shown in 1A. The expected bands at 8.5 and 15.7 kb were obtained. (C) Conditional mutant mice. Right part shows the Oprm1 ^fl conditional allele and excised allele (deletion of exons 2 & 3) after intercrossing Oprm1 ^fl/fl mice with Nav1.8-Cre mice. A and B indicate PCR primers used to detect gene excision, and C & D PCR primers for the floxed allele. PCR shows exon 2-3 deletion in DRGs but not brain of mu-cKO mice. In DRGs, the two bands result from gene excision in Nav1.8⁺ neurons but not in other Nav1.8-negative cells. Mu-KO mice show full deletion in both DRGs and brain. (D) Conditional knockout of mu opioid receptor gene in DRGs but not brain. Quantitative RT-PCR was used to measure Oprm1 mRNA levels from mu^fl, mu-cKO and mu-KO mice. Oprm1 mRNA expression was normalized to mu^fl control samples, and is decreased in mu-cKO animals. Oprm1 transcripts were undetectable in DRG and brain from mu-KO animals. ★★ P<0.01, mu-cKO vs mu^fl controls. (E) Conditional KO of the mu opioid receptor gene occurs in small/medium DRG cells. Left, representative in situ hybridization on DRG sections from mu^fl, mu-cKO and mu-KO mice. Thin, medium and large arrows point to small, medium and large cells, respectively. Scale bar = 100 µm. Right, cell size distribution of Oprm1-positive neurons in DRGs. The % of Oprm1-positive neurons in control and mu-cKO DRGs are shown in white and black, respectively. The % of Oprm1-positive cells is significantly reduced in small and medium, but not large diameter (>700 µm²) neurons from mu-cKO mice. ★★★ P<0.001 mu-cKO vs mu^fl controls, Student t-test.