Methods

Construction of an Adam11 gene-targeting vector
The 13.7-kb BamHI DNA fragment covering exons 2 – 20 of the Adam11 gene was amplified from C57BL/6 genomic DNA by the PCR using Expand Hi-Fidelity polymerase mix (Roche Diagnostics KK, Tokyo, Japan). To generate a mutation in the mouse Adam11 gene, we inserted a PGKneo cassette into exons 5 – 7 of the Adam11 gene. The final targeting construct consisted of a 10.1-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 [39].

Generation of Adam11 deficient mice
The linearised targeting vector was electroporated into TT2 embryonic stem (ES) cells [40]. Homologous recombinants were selected by PCR using the following primers, AGN1: 5'-TCGTGCTTTACGGTATCGCCGCTCCCGATT-3' in the PGKneo cassette, and SGN033: 5'-GGACCCGGAAGACATTTGCACGGT-3' outside the targeting vector. Homologous recombined DNA was efficiently amplified by 35 cycles of the following amplification steps (94°C-30 sec and 68°C-5 min). Correctly targeted ES clones were injected into fertilised ICR mouse eggs at the eight-cell stage. The resulting male chimeras were mated with C57BL/6NCrj females (Charles River Japan, Tokyo, Japan), and resulting heterozygous (+/-) male and female mice were interbred to generate homozygous (-/-) mice.

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 BE2K probe, which was a 2.0-kb BamHI – EcoRI genomic fragment located in intron 2.

Western blot analysis
The absence of ADAM11 protein in the (-/-) mice was confirmed by Western blot analysis. To be brief, the cerebellum was isolated from a six month-old mouse of each genotype and homogenised in cell lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1% NP-40, protease inhibitor cocktail [Roche Diagnostics]). After removal of cell debris by centrifugation, the glycosylated proteins in the supernatant were concentrated by the affinity chromatography using Con A Sepharose 4B (Amersham Biosciences Corp.). Each sample was separated on 10% SDS-PAGE, and transferred to a PVDF membrane. The blot was then incubated with monoclonal antibody against the ADAM11 (U. S. Patent 5,631,351) at 1 μg/ml. Bound antibodies were visualised with horseradish peroxidase-labelled second antibody and an ECL-plus chemiluminescence detection system (Amersham Biosciences Corp.).

Behavioural analysis
All animal procedures conformed to the Japanese regulations on animal care and use, following the Guideline for Animal Experimentation of the Japanese Association for Laboratory of Animal Science, and were approved by the Animal Care and Use Committee of Eisai Co., Ltd. For the present experiments, ADAM11-deficient male mice (backcrossed generations into C57BL/6NCrj, n = 14) were used in all behavioural analyses (22–24 weeks of age). All of the behavioural studies were conducted between 10:00 and 16:00 by a well-trained experimenter blind to the mouse genotypes.

Spontaneous motor activity
Locomotor activity in a novel environment was measured by introducing mice (+/+, +/- and -/-; n = 8, 8 and 8, respectively) for 90 min in a Plexiglas box (30 × 20 × 13 cm) using a VERSAMAX equipment and software (Accuscan, Columbus, OH, USA).

Water maze task
Spatial learning was assessed by three variants of the Morris water maze task [27] adapted for mice. Hidden platform task: The pool was divided into four quadrants with four starting locations, called north, east, south and west, at equal distances to the rim. A circular, transparent acrylic platform (diameter 8 cm) was submerged 1 cm below the surface of the water in the centre of the southeast quadrant for each trial of this task. Each mouse was allowed to swim for 60 sec and the time required to reach the platform (escape latency) was recorded in each trial. In total this task consisted of four trials per day over 9 days (1 min inter-trial intervals). Each trial was initiated by randomly placing an animal in one of the four starting locations. Probe trial: A single probe trial was carried out after the hidden platform task had been completed. In this trial, the platform was removed and the movement of each mouse was monitored using a computer-based video tracking system (BTA-2; Muromachi Kikai Co., Ltd., Tokyo, Japan). Each mouse was placed into the northwest quadrant of the pool and allowed to swim for 60 sec. Swim path length and the time spent in the trained (southeast) quadrant were calculated. Visible platform task: In this task, the circular platform was made visible by attaching a black board to it and the mouse was required to locate the visible platform. The mouse was allowed to swim for 60 sec and this task consisted of four trials per day for three consecutive days (1 min inter-trial intervals). Each trial was initiated by randomly placing an animal in one of the four starting locations.

Rotating rod test
Motor coordination was assessed with a rotating rod apparatus (KN-75, Natsume Seisakujo, Tokyo, Japan), which consisted of a plastic rod (3 cm diameter, 8 cm long) with a gritted surface flanked by two large discs (40 cm diameter). The mice were first placed on the stationary rod for 4 successive trials, followed by 4 trials at a rotation speed of 5 rpm, 8 trials at 10 rpm, and 20 trials at 15 rpm in that order. Latency until a fall occurred was monitored for 120 sec. Intra-trial intervals for each animal were more than 20 min.

Traction test
The grip strength of each mouse was measured with a traction apparatus (FU-1, Muromachi Kikai Co., Ltd.). Each mouse was made to grasp the attached bar (2 mm diameter) with the forepaws and was slowly pulled back by the tail. The maximum tension before release was recorded.

Wire suspension test
A mouse was placed on a stainless bar (50 cm length, 2 mm diameter, elevated up to 37 cm from a surface) at a point midway between the supports and observed for 30 sec in four separate trials. The amount of time spent hanging was recorded and scored according to the following system [41]: 0, fell off; 1, hung onto the bar with two forepaws; 2, in addition to 1, attempted to climb onto the bar; 3, hung onto the bar with two forepaws and one or both hind paws; 4, hung onto the bar with all four paws with tail wrapped around the bar; 5, escaped to one of the supports.

Footprint test
Black ink was applied to the hind paws of each mouse and the mice were placed in a narrow alley (9 × 25 × 10 cm) on white paper. Stride length and step width were calculated.

Statistics
Data are expressed as the means ± SEM. Statistical analysis was conducted using the software package SAS 8.1 (SAS Institute Japan Ltd., Tokyo, Japan). Statistical significance was determined by analysis of variance (ANOVA) followed by the Dunnett-type multiple comparison test, and p values of less than 0.05 were considered to be significant in the water maze task. Behavioural data in the rotating rod test were analysed by one- or two-way ANOVA with repeated measures, and p values of less than 0.05 were considered to be significant.

Histopathology
Sections of tissues including the brain, spinal cord, heart, lung, liver, kidney, spleen and testis from mice (+/+, +/- and -/-; n = 7, 7 and 7, respectively) were fixed in 10% buffered formalin and embedded in paraffin. Each paraffin section of thick 4 μm was stained with hematoxylin and eosin (HE).