A laboratory strain B6 was purchased from CLEA Japan Inc. (Tokyo, Japan), while nine field strains (BFM/2, BLG2, CHD, HMI, KJR, MSM, NJL, PGN2 and SWN) were selected in NIG (Shizuoka, Japan) (Table 1). All mice were maintained at the animal facility in NIG, under specific pathogen-free conditions and under controlled lighting conditions (daily light period: 06:00 to 18:00). Mice were kept in a temperature-controlled room (23°C ± 2°C) and food and water were provided ad libitum.
In the egg transfer experiments, B6C3F1 females were obtained through crosses between B6 female mice and C3H/HeNJcl male mice (CLEA Japan Inc.), and then used to serve as pseudopregnant recipients which were generated after mating with vasectomized male ICR mice (CLEA Japan Inc.).
Unfertilized eggs for IVF experiments were collected from female mice (over 8 weeks old) of one laboratory strain (B6) and nine wild strains. In the IVF experiments for each strain, 10 females were used in total and the IVF operations were performed twice, with five females each. Female mice were superovulated by means of an intraperitoneal injection of 5 IU of pregnant mare serum gonadotropin (Serotropin, ASKA Animal Health, Tokyo, Japan), followed 48 h later by an injection of 5 IU of human chorionic gonadotropin (Gonatropin, ASKA Animal Health). Approximately 17 h after gonatropin injection, eggs were removed from the ampulla of the oviduct and incubated in human tubal fluid (HTF, ARK Resource, Kumamoto, Japan), at 37°C, in an atmosphere containing 5% CO2. In each experiment, oocytes were collected from five superovulated females in each strain and all oocytes were incubated in medium, followed by counting the number of oocytes for five females each time. Sperm cells were obtained from male mice (over 9 weeks old) of the same strain from which the eggs were collected and incubated in HTF medium, at 37°C for 1 h. Immediately after obtaining the eggs, the sperm were introduced into a drop of HTF medium containing eggs, and the drop of HTF medium was incubated at 37°C, in a 5% CO atmosphere.2. Four to five hours after insemination, fertilized eggs were moved to a fresh drop of HTF medium and washed to remove cumulus cells and sperm by pipetting. After washing, fertilized eggs were moved to drops of potassium-supplemented simplex optimized medium (KSOM, ARK Resource) and incubated at 37°C in a 5% CO atmosphere.2, overnight. Normally developed 2-cell embryos, abnormal cells, and unfertilized eggs were observed and documented using the camera in an inverted microscope (IX71, Olympus, Tokyo, Japan), after which the 2-cell embryos were were transferred into the oviducts of pseudo-pregnant B6C3F1 females. .
CRISPR gRNAs, which were used to perform genome editing using the CRISPR/Cas9 system on the Adcyap1 gene encoding the neuropeptide PACAP, were engineered using CRISPOR43, followed by the CRISPR-Cas9 guide RNA design checker (https://sg.idtdna.com/site/order/designtool/index/CRISPR_SEQUENCE) developed by Integrated DNA Technologies (Coralville, IA, USA). No polymorphisms in the nine wild-type and B6 strains were found in gRNA sequences designed by MoG+, High Value Mouse Genome Database (https://molossinus.brc.riken.jp/mogplus /#JF1). gRNAs were generated as follows:
STEP 1 The Cas9 tracrRNA scaffold DNA fragment was PCR amplified with a primer set (gRNA-F/T7-gRNA-PCR-R, Supplemental Materials S1), using Tks Gflex DNA Polymerase (Takara Bio, Shiga, Japan) and plasmid DR274 (plasmid number 42250, Addgene, Watertown, MA, USA) linearized using DraI (Nippon Gene, Tokyo, Japan) as a model.
2ND STEP Two DNA fragments containing the T7 promoter and gRNA scaffold, PACAP-ex3-52fw and PACAP-ex3-56fw, were synthesized using the purified PCR product amplified in STEP 1 as template and KOD- Plus-Neo (Toyobo, Osaka, Japan) plus primer sets, PACAP-ex3-52fw/T7-gRNA-PCR-R and PACAP-ex3-56fw/T7-gRNA-PCR-R, respectively (supplementary material S1). The two DNA fragments used for the synthesis of the guide RNA were purified using the FastGene® Gel/PCR extraction kit (Nippon Genetics, Tokyo, Japan).
STEP 3 Two gRNAs were transcribed in vitro using two purified DNA fragments as templates, with the MEGAshortscript™ T7 transcription kit (Thermo Fisher Scientific, Waltham, MA, USA). The two gRNAs were purified using the MEGAclear™ kit (Thermo Fisher Scientific).
All primers were commercially synthesized by Thermo Fisher Scientific. The ssODN, PACAP_Larm-3xSTOP-spA-Rarm, which has three stop codons in three coding frames followed by a poly-A signal was commercially synthesized by Eurofins Genomics (Tokyo, Japan) and used as a template for repair homology-directed, to generate mice carrying the modified gene for PACAP (Supplementary Material S3).
Preparation of the electroporation solution
The electroporation solution contained two gRNAs synthesized in vitro, a commercially purchased Alt-R Sp Cas9 Nuclease V3 protein (Integrated DNA Technologies) and an ssODN. The final component concentrations were 1 µg/µl (Cas9 protein), 1 µg/µl (each gRNA) and 2 µg/µl (ssODN). The solution was diluted using Opti-MEM (Thermo Fisher Scientific) to 3 μl per subject female mouse. These components were mixed on ice. The solution was heated at 37°C for 10 min before use.
Genome editing using I-The GONAD method was conducted based on previous reports33.36. Female mice older than 8 weeks were co-housed with adult male mice of the same strain, at a time between 4:00 p.m. and 6:00 p.m., and kept overnight. Copulation plugs were confirmed by visual inspection the next morning between 9:00 and 10:00 a.m. Females that exhibited a vaginal plug were used for electroporation experiments on the same day. The surgeries were performed between 3:00 p.m. and 6:00 p.m. Females that exhibited a plug during copulation were anesthetized with 2-4% isoflurane (Pfizer Japan, Tokyo, Japan) using an inhalation anesthesia system (Narcobit-E II , Natsume Seisakusho, Tokyo, Japan) and placed on a hot plate at 37°C. Their dorsal skin was cut with scissors, after which the ovary, oviduct and part of the uterus were exposed outside the dorsal skin. Approximately 1.0 to 1.5 µl of electroporation solution was injected into the lumen of the oviduct upstream of the ampulla of the oviduct, using a micropipette, under observation under a stereomicroscope (SZX7 , Olympus). After injection, the oviduct and its ampulla were covered with a piece of wet paper (Kimwipe, Nippon Paper Crecia, Tokyo, Japan) dipped in phosphate-buffered saline (PBS) and grasped with forceps-type electrodes. depilate (CUY652P 2.5×4, Nepa Gene, Chiba, Japan) soaked in PBS. Electroporation was performed using the NEPA21 electroporator (Nepa Gene). The electroporation parameters were as follows: Poring pulse: 40 V, 5 ms pulse, 50 ms pulse interval, 3 pulses, 10% decay, ± polarity and transfer pulse: 10 V, 50 pulse ms, 50 ms pulse pulse interval, 6 pulses, 40% decay, ± polarity. Field strains that exhibited a low pregnancy rate (I-GONAD experiments with modified electroporation pulses, from 6 to 3, in terms of transfer pulses. No other parameters were changed in this experiment. After electroporation, the ovary, oviduct and part of the uterus were put back under the skin and the severed site was sutured using Wound Clip (Becton Dickinson, Franklin Lakes, NJ, USA United). After 20 days, the submitted mice were checked for the presence of fetuses.
i-GONAD Efficacy Analysis
Genomic DNA was extracted from an ear sample from each progeny. Each atrium was placed in a 1.5 ml tube with 100 µl of TE buffer (1 M Tris-Hcl, 0.5 M ethylenediaminetetraacetic acid (EDTA), pH 8.0), and the sample tube was heated to 95°C for 5 min. Once the sample had returned to room temperature, 2 µl of 10 mg/ml proteinase K (Roche, Basel, Switzerland) was added to each tube and incubated overnight at 55°C. After the incubation, the samples were heated at 95°C for 5 min and the solutions were directly used for PCR amplification, as template DNA. PCR amplification for target loci was performed in 10 µl total solution, containing 5 µl of 2× buffer for KOD FX Neo (Toyobo), 2 µl of 2 mM deoxyribonucleoside triphosphates (dNTPs), 0.3 μl of each primer (PACAP-ex3-left F1 and PACAP-ex3-right R1) (supplementary material S1), 0.05 μl of KOD FX Neo, 1.35 μl of H2O, and 1 μl of template DNA, using the following conditions: denaturation at 94°C for 2 min, 35 cycles of 98°C for 10 s, 60°C for 30 s, 68°C for 30 s and a extension at 68°C for 1 min. DNA products were purified using the FastGene® Gel/PCR extraction kit (Nippon Genetics). Purified DNA products were examined by 3% agarose gel electrophoresis, after which amplicons with mutant alleles were sequenced. Sequence analyzes helped clarify whether genome editing sites were precisely introduced or mutated with unexpected sequence changes. The sequences of the PCR products were generated by the Eurofins sequencing service.
Graphs and figures
All graphics and illustrations were generated using Prism 9 (GraphPad Software Inc., San Diego, CA, USA) and BioRender (https://biorender.com), respectively.
Institutional Review Board Statement
The study was conducted in accordance with ARRIVE guidelines and institutional guidelines, and was approved by the NIG Institutional Animal Care and Use Committee (permit numbers 31-7, 31-8, R2 -13 and R2-15).