Animals
Animals were housed under a 12 h light/dark cycle (light on at 8:00, off at 20:00) with maintained temperature (23 ± 1°C) and humidity (55 ± 10%) and ad libitum access to food and water. Animal experiments were approved by the Institutional Animal Care and Use Committee of The University of Tokyo and conducted in accordance with the guideline of The University of Tokyo.
Preparation of single-guide RNAs (sgRNAs), Cas9 mRNA and single-stranded oligodeoxyribonucleotide (ssODN)
To design sgRNA with smaller number of off-target sites, CRISPRdirect software was used (http://crispr.dbcls.jp; [28]). Two pairs of sgRNAs positioned on either endpoint of the deletion were designed. The protospacer sequences of sgRNAs were listed in Additional file 1 (Supplementary Table 1). These protospacers were cloned into BsaI-pDR274 vector (Addgene #42250). The sgRNAs were transcribed in vitro using the DraI-digested pDR274 vectors as a template and the MEGAshortscript T7 kit (Ambion, CA, USA) and purified using MEGAclear kit (Ambion). The Cas9 mRNA was transcribed in vitro using a MessageMax T7 ARCA-Capped Message mRNA transcription kit (Cellscript, WI, USA), polyadenylated with an A-plus Poly(A) Polymerase Tailing kit (Cellscript) and purified using MEGAclear kit (Ambion). The ssODNs designed to bridge the deletion endpoints were 120 nucleotides in length and positioned directly adjacent to the most external sgRNA site. The sequences of ssODNs were listed in Additional file 2 (Supplementary Table S2).
Microinjection of hCas9 mRNA, sgRNAs and ssODN
Cas9 mRNA, sgRNAs and ssODN were delivered by microinjection to the cytoplasm of C57BL/6N (CLEA Japan Inc., Tokyo, Japan) fertilized eggs as described previously [21]. Briefly, fifty ng/μL of hCas9 mRNA, 25 ng/μL of sgRNA (each) and 100 ng/μL of ssODN were mixed in RNase-free water and microinjected. Survived microinjected embryos were cultured in modified Whitten’s medium (mWM) until they reached the 2-cell stage. Injected embryos were transferred into oviducts of 0.5-day-post-coitum recipients (ICR, CLEA Japan Inc.).
Genotyping of genome engineered mice by PCR assay
Genomic DNA was extracted from mouse tail tips to serve as templates for genotyping PCR assay. PCR products were amplified with Ex Taq DNA polymerase (Takara Bio Inc., Shiga, Japan). The PCR primers for genotyping of Del(1.4Mb)/+ mice were as follows: 5’-AACCACAGGGGTGGAAAGTC-3’ and 5’-TGCTAGCCTGCATCGTAAGG-3’ (552 bp product, the deletion band); 5’-CACTTGTCAACTGACTACTGTTTG-3’ and 5’-GAGCCAGGTCTAGGAACGTC-3’ (654 bp product, the internal control). The following primers were used for genotyping of Del(1.5Mb)/+ mice: 5’-CTAAGGAATGGTTCCGGCCA-3’ and 5’-TTTCACGGAGGCGGTATTCA-3’ (424 bp product, the deletion band); 5’-GAGAAAGTGGGTGGGAAGGC-3’ and 5’-GTCCCTCGCCACAGTCATAA-3’ (532 bp product, the internal control). The reaction for detection of 1.4-Mb deletion was conducted under the following conditions: initial denaturation at 98°C for 2 min, 30 cycles of melting at 98°C for 30 s, annealing at 64°C for 30 s, and extension at 72°C for 30 s, with additional extension at 72°C for 2 min at the end. The condition of 1.5-Mb deletion was as follows: 98°C for 2 min, 30 cycles of 98°C for 30 s, 65°C for 30 s, and 72°C for 30 s, with additional extension at 72°C for 2 min at the end. PCR products were analyzed in 2% agarose gel electrophoresis and the sequences were confirmed by Sanger sequencing (Fasmac Co., Kanagawa, Japan).
Array comparative genomic hybridization (array CGH) analysis
Array CGH analysis was performed according to previously reported methods [12]. In briefly, an Agilent SurePrint G3 Mouse CGH 4x180K Microarray (Agilent, Santa Clara, CA, USA) was used according to the manufacturer’s instructions. Copy number variation (CNV) calls were made with Nexus Copy Number software v9.0 (BioDiscovery, El Segundo, CA, USA) using the Fast Adaptive States Segmentation Technique 2 (FASST2) algorithm, which is a hidden Markov model-based approach. The log2 ratio threshold for copy number loss (or deletion) was set at −0.4. The significance threshold to adjust the sensitivity of the segmentation algorithm was set at 1 × 10−6, and at least five contiguous probes were required for CNV calls. Genomic locations are reported in NCBI Build 37/UCSC mm9 coordinates.
Quantitative RT-PCR
Total RNA was isolated from hippocampi of male mice using miRNeasy Mini Kit (QIAGEN, MD, USA). For quantification of mRNA, first strand cDNA was synthesized using PrimeScript RT reagent Kit (Perfect Real Time) (Takara Bio Inc.). Briefly, 300 ng of total RNA was reverse transcribed using 25 pmol of oligo dT primer and 50 pmol of random 6 mer in 10 μL reaction. The resultant cDNA was diluted at 1:30 ratio in TE. All samples within an experiment were reverse transcribed at the same time. All real-time PCR reactions were performed using the PowerUp SYBR Green Master Mix (Applied Biosystems, CA, USA) and the StepOnePlus Real-Time PCR system (Applied Biosystems). The experiments were conducted in triplicate for each data point. The relative quantification in gene expression was determined using ∆∆Ct method [29]. The sequences of the primers are listed in Additional file 3 (Supplementary Table 3).
Behavioral tests
All behavioral tests were carried out with male mice at the age of 8–11 weeks. We used Del(1.4Mb)/+ and Del(1.5Mb)/+ lines which were backcrossed to C57BL/6N for 3 generations. Del(3.0Mb)/+ mice was backcrossed to C57BL/6N for 4 generations. Prior to each experiment, mice were placed in the testing room for at least 1 h to acclimate to the experimental environments. The experimenter was blind to genotype throughout the experimental procedures. All apparatus used in behavioral tests were cleaned with 70% ethanol and wiped with paper towels between each session.
Open field test
Mice were placed in the center of the open field arena (diameter: 75 cm, height: 35 cm) and were allowed to explore freely the arena for the following 10 min under moderately light conditions (15 lux). Their movement was recorded with a camera mounted above the arena, and their activity was measured automatically using Smart V3.0 tracking software (Panlab, Barcelona, Spain). The open field was divided into an inner circle (diameter: 50 cm), and an outer area surrounding the inner circle. Measurements included total distance moved, time spent in the outer zone and number of transitions between inner and outer section.
Five-trial social interaction test
Five-trial direct social interaction test was performed as described previously [21]. Subject mice were placed individually into home cage (45 cm × 28 cm × 16 cm) for 1 h before starting test under the moderately light conditions (15 lux). A juvenile intruder mouse (5-week-old) was introduced into the subject mouse’s home cage. The subject mouse was exposed to the same intruder mouse for 5 min over 4 trials with an inter-trial-interval of 30 min. During the fifth trial, the subject mouse was exposed to a novel intruder mouse (5-week-old). The time spent in social interaction (close following, inspection, anogenital sniffing and other social body contacts) was recorded.
Prepulse inhibition (PPI)
The prepulse inhibition (PPI) test was carried out by using SR-Lab system (San Diego Instruments, San Diego, CA, USA) as described previously [21]. After each mouse was placed in an enclosure (12.7 cm, 3.8 cm inner diameter) under moderately bright light condition (180 lux), they were acclimated for 10 min in the presence of background white noise (65 dB). The movement of the animal in the startle chamber was measured by a piezoelectric accelerometer mounted under the enclosure at the sampling rate of 1 kHz. Individual mouse received 20 startle trials, 10 no-stimulus trials and 40 PPI trials. The inter-trial interval was between 10 and 20 s. The startle trial consisted of a single 120 dB white noise burst lasting 40 ms. PPI trials consisted of a prepulse (20 ms burst of white noise at 69, 73, 77 or 81 dB intensity) followed by the startle stimulus (120 dB, 40 ms white noise) 100 ms later. Each of the four prepulse trials (69, 73, 77 or 81 dB) was carried out 10 times. Five consecutive startle trials were presented at the beginning and end of the session. The remainder of sixty different trials were performed pseudorandomly to ensure that each trial was done 10 times and that no two consecutive trials were identical. During the session, 65 dB background white noise was continually present. The largest amplitude in the recording window was taken as the startle amplitude for the trial. Basal startle amplitude was determined as the mean amplitude of the 10 startle trials. PPI (%) was calculated as follows: 100 × (pulse-alone response − prepulse-pulse response)/pulse-alone response, in which prepulse-pulse response was the mean of the 10 PPI trials (69, 73, 77 or 81 dB) and pulse-alone response was the basal startle amplitude.
Drug administration
Haloperidol Solution (5.0 mg/mL) was obtained from Sumitomo Dainippon Pharma Co., Ltd. (Tokyo, Japan) and was diluted in saline. Haloperidol (0.3 mg/kg) was administrated intraperitoneally (i.p.) 30 min before PPI experiment.
Contextual and cued fear conditioning test
The fear conditioning test was conducted using ImageJ FZ1 (O’Hara & Co., Ltd., Tokyo, Japan) as described previously [21]. The conditioning chamber was a square arena (10 cm × 10 cm × 10 cm) with clear Plexiglas walls and a metal grid floor connected to a circuit board that delivered electric shocks to the metal grid. A video camera was set in front of the cage to record the behavior. In the conditioning session, mice were individually placed into the conditioning chamber and allowed to explore freely for 3 min. After 3 min exploratory period, each mouse was exposed to two tone-footshock pairings (tone, 30 sec; footshock, 2 sec, 0.8 mA at the termination of the tone; separated by 1 min intertrial interval). One min after the second footshock, the mouse was returned to its home cage. Twenty-four h after conditioning, the context-dependent test was performed, in which each mouse was placed back into the conditioning chamber, and the freezing response was measured for 6 min in the absence of the conditioned stimulus. Forty-eight h after the footshock, each mouse was tested for auditory (tone) fear conditioning in a novel opaque chamber. Different environmental cues (e.g. light condition and background noise) were provided in the novel chamber. Mice were tested in the novel chamber for a 3 min baseline period (pre-tone) followed by another 3 min for the conditioning tone during which the tone was presented persistently for 3 min. Total freezing rate was measured as an index of fear memory. Motionless bouts lasting more than 2 s were considered as freeze.
Electroencephalogram (EEG) and electromyogram (EMG) analysis
Sleep analysis was performed as described previously [30]. Eight- to ten-week-old mice were subjected to EEG/EMG electrode implantation surgery. The surgery was performed under isoflurane anesthesia (4% for induction, 2% for maintenance). The scalp was incised along the midline to expose the cranium. Four holes were generated on the skull using 1.0-mm drill bits (anteroposterior: 0.50 mm, lateral: ±1.27 mm and anteroposterior: −4.53 mm, lateral: ±1.27 mm from bregma). The four electrode pins were lowered onto the dura under stereotaxic control (David Kopf Instruments, #940/926) and fixed using dental cement (3M ESPE, Ketac Cem Aplicap). Subsequently, two EMG wires were inserted into the neck extensor muscles and covered with dental cement. A 6-pin header dissected from 2 x 40 pin header (Useconn Electronics Ltd., #PH-2x40SG) was inserted to the top of the EEG/EMG electrode into close the holes of the insulator.
Seven days after surgery, the mice were attached to a tether cable and singly housed in a recording cage (19.1 x 29.2 x 12.7 cm). The tether cable was hung by a counterbalanced lever arm (11.4 cm-long, Instech Laboratories, #MCLA) that allows the mice to move freely. All mice were allowed at least 5 days of recovery from surgery and habituation to the recording conditions for at least 4 days. The floor of the cage was covered with aspen chips and nest materials. To examine sleep/wake behavior under baseline conditions, the EEG/EMG signal was recorded for three consecutive days from the onset of the light phase.
EEG/EMG signals were amplified, filtered (EEG: 0.5–100 Hz; EMG: 0.5–300 Hz) with a multi-channel amplifier (NIHON KODEN, #AB-611J), and digitized at a sampling rate of 250 Hz using an analogue-to-digital converter (National Instruments, #PCI-6220) with LabVIEW software (National Instruments). The EEG/EMG data were visualized and semi-automatically analyzed by MATLAB-based software followed by visual inspection. Each 20-sec epoch was staged into wakefulness, non-rapid eye movement (NREM) sleep and REM sleep. Wakefulness was scored based on the presence of low amplitude, fast EEG activity and high amplitude, variable EMG activity. NREM sleep was characterized by high amplitude, delta (1–4 Hz)-frequency EEG waves and a low EMG tonus, whereas REM sleep was staged based on theta (6–9 Hz)-dominant EEG oscillations and EMG atonia. The total time spent in wakefulness, NREM sleep, and REM sleep were derived by summing the total number of 20-s epochs in each state. Mean episode durations were determined by dividing the total time spent in each state by the number of episodes of that state. Epochs that contained movement artifacts were included in the state totals but excluded from the subsequent spectral analysis. EEG signals were subjected to fast Fourier transform analysis from 1 to 30 Hz with 1-Hz bins using MATLAB-based custom software. The EEG power density in each frequency bin was normalized to the sum of 16-30 Hz in all sleep/wake state.
Statistical analysis
The significance of differences (p < 0.05) was assessed by two-tailed Welch’s t-test for comparison of two groups. In multiple comparison, the significance of differences was evaluated by an analysis of variance (ANOVA) with two-way repeated measures and Bonferroni post hoc analysis. All data are expressed as the mean ± standard error of the mean (SEM).