All tests were approved by the United Kingdom Home Office Animals (Scientific Procedures) Act 1986. Experiments were conducted using both male and female littermate mice, all of which were at least 6 weeks old when tested. The same observer performed all experiments and was blind to the genotype of the animals.
Generation of transgenic construct
To create the construct for production of the transgenic AvCreERT2 mouse line we used a recombineering technique . The CreERT2 containing plasmid which also contained a FRT flanked Kanamycin selection cassette was a gift from Prof Chambon .
Briefly, the start codon of Advillin is located in exon 2 of the gene. The 5' homologous arm of the construct (0.45 kb) was amplified from a BAC containing exon 2 of the Advillin gene and cloned into the AscI/SalI restriction sites of the CreERT2 vector. The 3'homologous arm (0.3 kb) was amplified from the same BAC and cloned into the PacI/EagI restriction sites of the CreERT2 vector. The completed shuttle construct was sequenced and the targeting cassette was isolated from the plasmid by AscI/PacI digest.
EL250 E. coli cells were transformed with the BAC containing the Advillin gene. Isolated BAC DNA from the individual clones was checked by restriction analysis followed by pulse-field gel electrophoresis. Only the clones displaying the correct pattern of the restriction fragments were used for the recombineering procedure. The correct bacterial clones were co-transformed by shuttle construct and recombination was induced by incubating the bacteria at 42°C for 15 minutes. The induced bacterial clones were isolated by growing them on Kanamycin containing plates. The Kanamycin resistance cassette was removed by inducing Flp-recombinase expression by arabinose. The final construct was verified by PCR and analytical restriction digests using NotI and PacI.
Transgenic mouse production and screening
The resulting BAC was isolated, digested by Not I, gel purified and used for the oocyte microinjection. The founders were screened by PCR and Southern blot. The primers for PCR are as follows:
AdF:GACAGATTATCTGCAATCTCTCTAAG, AdR:AGAGCACAGAGCCACCCTCGAGAC, CREF:GGCCTGGTCTGGCCACTCTGCCAG, CRER:GTTCCTGATGTCCTGGCATCTGTC.
The products of PCR reactions are AdF/AdR - 0.95 kb(wt) and 3.39(tg), AdF/CreR - 1.027 kb and CreF/AdR - 1.99 kb. The products were resolved on 1% agarose gel, stained with ethidium bromide and photographed.
Southern blot analysis
Genomic DNA was extracted from tail snips . The 5' probe which recognizes both the wildtype gene and transgene was amplified from BAC using the following primers Avil5HS: GACGGCGCGCCCTCAGGAATATG TGTTGCCTTTC; Avil5HAS: TCTGTCGACCATAGTGGCTGTCTTCCTGGAAC.
Southern blot analysis with HindIII digested genomic DNA produced a 7.4 kb wildtype band and a 3.6 kb transgene band (Figure 1C).
Tamoxifen (Sigma T5648) and 4-OHT (Sigma H7904) solutions were prepared according to a previously described protocol . Eight week old AvCreERT2/ROSA26LacZ mice were injected with 2 mg of tamoxifen (ip) daily for 5 consecutive days. Two days after the last injection mice were euthanized and DRG were collected for further analysis. To characterize Cre induction during embryonic development, pregnant AvCreERT2/ROSA26LacZ mice were injected with either 2 mg of tamoxifen or vehicle daily for 5 consecutive days starting at day E12.5. On day E18.5 the mice were euthanized and the embryos were collected for further analysis. All embryos were genotyped.
Cultured DRG neurons were treated with 4-OHT for 3 days (1 μM on the first day and 2 μM the following two days). The neurons were fixed and analyzed the day after the final treatment.
DRG cell culture
AvCreERT2/ROSA26LacZ animals were killed by inhalation of a rising concentration of CO2 followed by cervical dislocation, and 30-40 DRG were dissected from each. Ganglia were digested in collagenase (Type XI, 0.6 mg/ml, Sigma), dispase (3.0 mg/ml, Sigma), and glucose (1.8 mg/ml) in Ca2+, Mg2+ free PBS for 40 min prior to mechnical trituration. Cells were then resupended in Dulbecco's modified Eagle's medium (Gibco) containing 10% fetal bovine serum (Gibco), 10,000 i.u./ml penicillin-streptomycin (Gibco), and 100 ng/ml nerve growth factor (Sigma), and plated on 13-mm cover slips coated with poly-L-lysine.
The staining was performed as previously described . Briefly, following the tamoxifen treatment, the DRGs were isolated and immediately frozen in OCT (O.C.T. Compounds, BDH) on dry ice. 12 μm cryosections were dried at room temperature for 30 min and then fixed with 4% PBS-buffered paraformaldehyde solution for 10 min on ice. After three washes in PBS, sections were incubated for 30 min in 10% goat serum diluted in PBS containing 0.3% Triton X-100 (PBST), and for 1 h at room temperature in a 1:500 dilution of an anti peripherin monoclonal antibody (P5117, Sigma) and 1:200 dilution of an anti N-200 polyclonal antibody (N4142, Sigma). Following three washes in PBST, the sections were incubated for 1 h in a 1:500 dilution of Alexa Fluor 488 goat anti-mouse IgG (A-11017, Molecular Probes) and 1:1,000 dilution of Alexa Fluor594 goat anti-rabbit IgG (A-11037, Molecular Probes). After three washes in PBST, the sections were mounted in CITIFlour solution and analysed using a fluorescent microscope.
After fixation with 4% PBS-buffered paraformaldehyde or after immunohistochemistry, DRG sections were washed three times with PBS and then incubated overnight in X-gal solution at 35°C. For cultured DRG neurons, the slides were fixed with 2% PBS-buffered formaldehyde containing 0.25% glutaraldehyde for 15 min at room temperature, and then stained with X-gal solution at 35°C overnight. Sections or slides (cultured DRG neurons) were counterstained with 1% neutral red, dehydrated with ethanol, and cleared with Histo-ClearII and mounted with DePex mounting medium (BDH). The X-gal staining in freshly frozen sections is usually punctate in appearance, but homogeneous in perfused DRG sections and in cultured DRG neurons.
The staining of the E18.5 embryos was performed as follows. The embryos were isolated from pregnant females killed by CO2 inhalation and cervical dislocation. The embryos were placed immediately into 1:40 dilution of sodium pentobarbitone in PBS for 5 min at room temperature. This was followed by 3 washes with PBS and the embryos were skinned to allow better penetration of staining solutions. Tail biopsies were taken for genotyping. The embryos were fixed at room temperature for 2 hours, washed and left over night in the LacZ staining solution at room temperature. The next day embryos were washed with PBS and were cleared by incubating for 24 hours at 4°C in the following solutions: 70% ethanol, 100% methanol, fresh 100% methanol and benzyl benzoate:benzyl alcohol (2:1). Cleared embryos were photographed and analyzed.
The motor coordination of transgenic and wildtype mice was assessed using the Rotarod test  before testing nociceptive responses.
Thermal nociceptive thresholds were measured using the paw-withdrawal latency according to the method described by , with minor modifications. As well as the hot-plate test (50 & 55°C) originally described by  and later modified by . Behavioural responses to cooling (approx. 10-15°C) were assessed using the acetone test, as described by .
Mechanical nociceptive thresholds were measured using a modified version of the Randall-Selitto test  that applies pressure to the tail via a 3 mm2 blunt probe . Touch perception thresholds were measured using the up-down method for obtaining the 50% threshold using von Frey hairs as described by .