Methods

Construction of an Adam22 gene-targeting vector
The 18-kb genomic DNA fragments covering exons 5, 6, 7, 8 and 9 of the Adam22 gene were amplified from C57BL/6 genomic DNA by PCR using Expand Hi-Fidelity enzyme mix (Roche Diagnostics) and primers designed for each exon. To generate a mutation in the mouse Adam22 gene, we inserted the PGKneo cassette into exon 8 of the Adam22 gene. The final targeting construct consisted of a 10.5-kb genomic DNA fragment that was interrupted by the insertion of the PGKneo cassette and contained a MC1/TK cassette as a negative selection marker against random integration [5].

Generation of Adam22 deficient mice
The linearised targeting vector was electroporated into TT2 embryonic stem (ES) cells [31]. Homologous recombinants were selected by PCR. The extracted genomic DNA from each clone was amplified using the forward primer AGN2: 5'-GCCTGCTTGCCGAATATCATGGTGGAAAAT-3' in the PGKneo cassette, and the reverse primer MFP065R: 5'-ACTATTTCTGTGATGAGGGCACAGCATC-3' outside the targeting vector. Homologous recombined DNA was efficiently amplified by 35 cycles of the following amplification steps (94°C-30 s and 68°C-5 min). The targeting efficiency of this construct was about 4%. Correctly targeted ES clones were injected into fertilized ICR mouse eggs at the eight-cell stage. The resulting male chimeras were mated with C57BL/6N females, and heterozygous male and female mice were interbred to generate homozygous mice. The ataxic phenotypes of homozygous mice were observed in two independent lines.

Southern blot analysis
Mouse genomic DNA used for Southern blot analysis was extracted from the liver of each genotype. BamHI-digested genomic DNA was probed with the [32P]-labelled 0.4-kb SpeI-BamHI genomic fragment, which is located between exons 8 and 9.

Antibody production
His-tagged and MBP-fused recombinant protein containing cytoplasmic domain (cp) of human ADAM22 isoform 1 (position: 756–906, 151 amino acids in length) were produced in E. coli. His-tagged ADAM22-cp protein was purified in denatured condition using Ni-NTA resin (Qiagen GmbH) and dialysed in PBS. Precipitated protein was recovered and was mixed with Freund's complete adjuvant (Invitrogen), then, the mixture was used for immunization of rabbits. The antiserum raised against the His-tagged ADAM22-cp protein was incubated with MBP-fused ADAM22-cp protein coupled to Affi-Gel 10 beads (Bio-Rad). Beads with bound antibodies were washed in PBS, and the bound antibodies were eluted with 100 mM glycine, pH 3.0, and neutralized immediately. Using the affinity purified antibody, both human and mouse ADAM22 proteins were detected by Western blot analysis. However, unfortunately the antibody was not suitable for immunohistochemical analysis of mouse tissues.

Western blot analysis
The absence of ADAM22 protein in the Adam22 -/- mice was confirmed by Western blot analysis. Briefly, the cerebellum was isolated from a P14.5 mouse of each genotype and homogenized with a Polytron homogenizer in cell lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1% NP-40, Complete protease inhibitors [Roche Diagnostics]). After removal of cell debris by centrifugation, the supernatant was separated on 10% SDS-PAGE, and transferred to a nitrocellulose membrane. The blot was then incubated with polyclonal antibody against the cytoplasmic domain of ADAM22 (at 1:1,000). Bound antibodies were visualized with horseradish peroxidase-labelled second antibody and a ECL-plus chemiluminescence detection system (Amersham Biosciences Corp.).

Primary Schwann cell culture
Primary Schwann cells were prepared from 4-month-old C57BL/6 mice according to Manent's protocol [32] with minor modifications. Briefly, sciatic nerves were removed and incubated for 7 days in the pre-treatment medium, which consisted of D-MEM (high glucose) supplemented with 10% FCS, 50 mg/ml gentamicin (Invitrogen Corp.), 2.5 μg/ml fungizone (Invitrogen Corp.), 2 μM forskolin (EMD Biosciences Inc.) and 10 ng/ml recombinant heregulin-beta1 (R&D Systems). For dissociation, cultured sciatic nerves were cut into pieces and incubated at 37°C for 3 hours in Opti-MEM medium (Invitrogen Corp.) containing 130 U/ml collagenase type I (Invitrogen Corp.) and 0.4 U/ml dispase II (Roche Diagnostics). Dissociated cells were resuspended in the pre-treatment medium and plated on Poly-D-Lysine/Laminin coated plate (BD Biosciences). The purity of the cultured Schwann cells, as determined by indirect immunofluorescence analysis, approached 90 %.

RT-PCR analysis
Adult C57BL/6 male mice were used in this study. Total RNAs purified from the cerebellum, spinal cord, sciatic nerve, DRG and cultured Schwann cells using TRIzol (Invitrogen Corp.) and RNeasy kit (Qiagen GmbH) were analysed by RT-PCR using SuperScript II and random primer (Invitrogen Corp.), and PCR amplification (40 cycles; 94°C-30 s, 60°C-30 s and 68°C-5 min) with Expand Hi-Fidelity DNA polymerase (Roche Diagnostics) and ADAM22-specific primers. To detect splicing variants in the cytoplasmic domain, a forward primer was placed just upstream of the transmembrane domain and a reverse primer was set on the terminating exon. The primers used in this study were as follows. ISH04-forward: 5'-AACAGGCACTGGACAGGGGCTGAC-3' and ISH04-reverse: 5'-AATGGATGTCTCCCATAGCCTGGC-3'.

Histopathology
Mice were anesthetized with ethyl ether. Whole-body perfusion by 2% paraformaldehyde-glutaraldehyde solution followed by heparin-included saline was performed. Sciatic nerves, trigeminal nerves, brain and spinal cord were removed and fixed in 10% neutral-buffered formalin. The spinal cord and other nerve blocks were washed and postfixed with 2% osmium tetroxide, and were dehydrated in ethanol and equilibrated in Epon. Epon embedded semithin sections were stained with toluidine blue and were subjected to light microscopic examination. For electron microscopic analysis, thin sections were cut using an ultramicrotome, stained with 1.5% uranylacetate in 50% ethanol and 0.8% lead citrate, and analysed using electron microscope. All procedures were conducted according to the Eisai Animal Care Committee's guidelines.

Immunohistochemistry
Frozen sections were rinsed in 0.1% Triton X-100/PBS at room temperature for 1 hour, and were blocked in BLOCKACE solution (Dai-nippon). The following antibodies were incubated overnight in 0.1% BLOCKACE at 4°C:
monoclonal mouse anti-calbindin 28 K (Sigma, 1:200); monoclonal mouse anti-MBP (SMI-99, Sternberger Monoclonals Inc., 1:50); monoclonal mouse anti-Neu N (CHEMICON, 1:100); rabbit anti-S100 polyclonal (DAKO, 1:200). Nuclei were counterstained with 1 μg/ml DAPI (Sigma). MBP staining was performed after microwave epitope retrieval in 10 mmol/L citrate buffer (pH 6). Sections were incubated for 1 hour with secondary antibodies: Cy3-labeled donkey anti-rabbit IgG (Jackson Immuno Research, 1:200), FITC-labelled donkey anti-mouse IgG (Jackson Immuno Research, 1:200). Sections were photographed with an Olympus microscope (Olympus IX71) equipped with a high-resolution CCD camera.

RNA in situ hybridisation
In situ hybridisation on frozen sections were carried out using 35S-labelled RNA probes. Briefly, 610 bp of mouse ADAM22 cDNA (position: 1351–1960 from initiating ATG) was cloned into the pBluescript II SK(+) or the pBluescript II KS(+) vector. Using these plasmids as templates, sense and antisense labelled RNA probes were generated by T7 RNA polymerase and [α35S]UTP. Frozen brain and spinal cord from 2 month-old C57BL/6 female mice were used in this analysis. Pretreatment, hybridisation, RNase treatment and washing was carried out following the protocol described in the literature [33]. Dehydrated slides were attached to imaging plates for 48 hours and the autoradiograms were analysed using a Bio-Image Analyser (BAS3000, Fuji Photo Film).