5.1. Animals and genotyping

All animals were housed in groups of 2–4 in a 14:10 h light/dark cycle (lights on at 7:00 am) and an ambient temperature of 22 ± 2 °C. Mice had access to standard chow and water ad libitum. Procedures were carried out in accordance with the U.K. Animals (Scientific Procedures) Act 1986 and associated guidelines.

To study changes in nociception and mRNA expression after SNI, 11–14 week old male 129/OlaHsd mice (Bristol University colony) were anesthetized, and the common peroneal and sural branches of the right sciatic nerve were sectioned leaving the tibial branch intact (Holmes et al., 2008). 7 days later mice were killed by cervical dislocation to obtain ipsilateral (SNI) and contralateral (control) lumbar L4 and L5 DRG pools each from 8 to 12 animals (n = 4). Tissues were frozen on dry ice and stored at − 80 °C.

Heterozygous mice with one targeted allele of Gpr151 were generated by Deltagen Inc. and obtained from the Jackson Laboratory (stock number 005805; strain B6.129P2-Gpr151^tm1Dgen/J, i.e. targeted mutation 1, Deltagen), in which a portion of the Gpr151 coding region (nt 155–271 of {"type":"entrez-nucleotide","attrs":{"text":"NM_181543","term_id":"1560941415","term_text":"NM_181543"}}NM_181543) was replaced by a SA-IRES-LacZ-Neo cassette, resulting in an allele with β-galactosidase expression under the control of the endogenous Gpr151 promoter and targeted disruption of the Gpr151 (Gpr151 MUT). All experiments were performed using 10–12 week old animals bred to homozygosity for the targeted allele and strain-, age- and sex-matched WT controls. Animals were genotyped by PCR with three primers:

Construct forward 5′-CTGGATCTCGAGTGATCAGGTACC-3′,

Gpr151 forward 5′- CCTAAACAAGAAGCTACCATCTGCA-3′,

Gpr151 reverse 5′-AGTCAGAGGACTTGCAGATGAACC-3′.

Cycling conditions were 95 °C for 7 mins; 40 cycles of 94 °C for 30 s/62 °C for 30 s/72 °C for 90 s; and a final 72 °C incubation for 10 min, resulting in amplified products of 178 and 395 bp from Gpr151 MUT and WT alleles, respectively.