1696 A. S. St u¨rzebecher and others J Physiol 588.10 the threshold currents (Matsutomi et al. 2006). Small molecules that speciﬁcally block the function of these VGSC subtypes include chemical tools (Jarvis et al. 2007), small interfering RNAs (Dong et al. 2007), and venom-derived toxins (Terlau & Olivera, 2004). The μO-conotoxins MrVIa and MrVIb were the ﬁrst group of peptide toxins reported to inhibit VGSC currents in mammalian dorsal root ganglia (DRG) neurones (Daly et al. 2004). MrVIa was found to inhibit TTX-R VGSC currents with an IC50 value of ∼80 nM and a ∼10 times higher IC50 value (∼1 μM) for TTX-S sodium currents (Daly et al. 2004; see Supplemental material associated with the current paper, Table 1, available online only). In this study we have used the tethered toxin approach (Iban˜ez-Tallon et al. 2004; Holford et al. 2009) combined with cell-speciﬁc transgenesis, to deliver a genetically encoded tethered form of the neurotoxin MrVIa to nociceptors in mice. We show that this approach can be successfully used to manipulate VGSC currents in a cell-autonomous manner. Furthermore, the nociceptor-speciﬁc inhibition of VGSC currents in these transgenic mice leads to speciﬁc changes in the ﬁring of noxious cold-sensitive nociceptors and reduction in inﬂammation-induced pain behaviour. Methods Mice were housed in the animal facility of the Max-Delbr u¨ck Center with ad libitum access to food and water in an air-conditioned room at 22–23◦C with a standard 12 h light/dark cycle. Mice were killed by placement in a CO2-ﬁlled chamber for a 2–4 min followed by cervical dislocation. All procedures conformed to the German guidelines of animal experimentation laid down by the government. Animal housing and care, as well as protocols for killing mice, are registered with and approved by the appropriate German federal authorities (Landesamt f u¨r Gesundheit und Soziales), which also governed proper implementation. Generation of Tg-t-MrVIa bacterial artiﬁcial chromosome (BAC) transgenic mice The mouse BAC clone (RP23-214H2) encompassing the Scn10a gene (Invitrogen) was modiﬁed to include the t-MrVIa expression cassette using the two-step recombination system (Gong et al. 2002). After electro- poration and homologous recombination, resolved clones were screened by PCR and Southern blotting. The modiﬁed BAC for injection was puriﬁed by CsCl, linearised and 3 ng μl−1 of BAC DNA was injected into fertilised oocytes of hybrid BDF mice. Southern blot analyses Genomic DNA (5 μg) from each transgenic founder line was digested with NcoI and separated by gel electrophoresis. After gel denaturation (0.5 M NaOH, 1.5 M NaCl) and equilibration (10 × saline–sodium citrate buffer (SSC)), membrane blotting (Hybond N+ from Amersham) was performed in 10 × SSC. The membrane was quickly washed in 2 × SSC, dried for 2 h and crosslinked with UV light (Hoefer). DNA probes were radioactively labelled with α-32P-dCTP (NEN) using the Prime-It RmT Random Primer Labeling kit (Stratagene). Twenty-ﬁve nanograms of the probe was puriﬁed via Probe-Quant G50 micro-columns (Amersham) and denatured at 95◦C for 5 min before hybridisation. Hybridisation was performed at 65◦C over- night in 5 × Denhardt’s solution, 6 × SSC, 0.5% SDS and 100 μg ml−1 salmon sperm DNA. The membrane was washed in 2 × SSC/0.1% SDS followed by stringent washes in 0.1 × SSC/0.1% SDS. Autoradiograms were exposed at −70◦C for 3 days. Reverse Transcription PCR (RT-PCR) Mouse tissues were rapidly dissected, collected in Trizol and homogenised. Total RNA was extracted by phenol/chloroform and treated with DNaseI (Invitrogen). RT-PCRs were performed using one-step platinum RT-PCR (Invitrogen). Primer pairs used were: t-MrVIa-F: 5 -TGGGAGTACTGCATAGTGCCGAT-3 , t-MrVIa-R: 5 -CCCGTAGTTCCATCCTTCCTTCAA-3 ; Nav1.8-F: 5 -TAACGTGTGGGTCTCTGTGC-3 , Nav1.8-R: 5 -AGGTATGGAGCCAGGTCCTC-3 ; β-actin-F: 5 -TCGTGCGTGACATCAAAGAGAAGC-3 , β-actin-R: 5 -ATGGATGCCACAGGATTCCATACC-3 . In situ hybridisation RNA probes were synthesised with T7 RNA polymerase using DIG labelling (Roche) and puriﬁed with the RNeasy kit (Qiagen). Dorsal root ganglia (DRG) from neonatal mice were collected in ice-cold PBS and frozen in OCT (Sakura). Cryosections were cut with 12 μm thickness. In situ hybridisation was performed with DIG-labelled probes as described (Muller et al. 2005). Patch-clamp electrophysiology and data analysis Whole-cell patch-clamp recordings were performed on DRG cultures from P7–P15 mice as previously described (Hu & Lewin, 2006) and 100 ng ml−1 nerve growth factor was added to the culture medium. Recordings were done 4–24 h after plating, using borosilicate capillaries with a resistance of 5–8 M . For VGSC recordings, cells were C 2010 The Authors. Journal compilation C 2010 The Physiological Society