To get the mutant Ccnb3 mice, the sgRNA 1 and 2 specifically tagerted the exon 1 of Ccnb3 and Cas9 nickase mRNA were injected to the fertilized eggs with standard procedure. Mouse Genome DNA was collected from the 1 mm tail of mouse using lysis buffer (add 50 mM NaOH 180ul, boil in water for 40 min and then add 20ul 1 M Tris-Hcl pH 8.0, get 5ul as temple for the PCR). Genotypes were identified by PCR analysis using primers F1: 5’-GTGAGGTAGCTGAAGCCTAT-3′ and R1: 5’-GATAGAACCTAAGGCTTGCC -3′. The wild type groups got a band at 560 bp, while the mutant groups got a band at 416 bp. Mouse Ccnb3 gene is locate in X chromosomes, therefore in male mice only one copy of Ccnb3 and in female mice there are two copy of Ccnb3.
The mice used in this study, which mixed backgrounds of 129 and C57BL/6 J, were obtained from the Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing China. All animals were kept in accordance with the protocols approved by the guidelines of the Institutional Animal Care and Use Committee of the Institute of Zoology (IOZ), Chinese Academy of Sciences (CAS), Beijing, China. After the experimentation, the animals used in this study were handled by the Laboratory Animal Center of the Institute of Zoology (IOZ), Chinese Academy of Sciences (CAS) according to the guidelines of the Institutional Animal Care and Use Committee of the Institute of Zoology (IOZ), Chinese Academy of Sciences (CAS), Beijing, China. Our experiments in this study were adhered to the ARRIVE guidelines (Additional file 2).
Total RNA isolation and RT-PCR analysis
Total RNA was extracted from adult testes using Trizol (TIANGEN, DP405–2, Beijing, China) according to the manufacturer’s protocol. Then, the total RNA concentration and purity were quantified using Nanodrop 2000 Spectrophotometer (Biolab, Scoresby, Vic., Australia). Before the reverse-transcribed of RNA, the genome DNA was cleared with the RNase-free DNase H. The RNA was then reverse-transcribed with 5× FastKing-RT SuperMix kit (TIANGEN, KR118–01, Beijing, China) according to the manufacturer’s protocols. We then use the Gapdh primers (For: 5′- ATGGTGAAGGTCGGTGTGAA-3’ Rev.: 5′- GCAGTGATGGCATGGACTGT-3′, 542 bp) and Ccnb3 primers (F2: 5′- CCACCACCACTACTACCCAA-3′ R2: 5′- GGCTTGTTGGGTATATCCAG-3′, 368 bp product for wild type) to amplify Gapdh and Ccnb3 and running the gel.
Western blot analysis
Protein was extracted from the adult mouse testes and then separated on 10% SDS-PAGE gels. The protein in PAGE gels was transferred to PVDF membrane and then blocked with 5% nonfat milk (in 1X PBS). The membranes were then probed with primary antibodies CCNB3 (Invitrogen, PA5–37254, Rabbit, 1:1000, California, USA) or GAPDH (Bioworld, MB001, Mouse, 1:5000, Minnesota, USA) overnight. The membrane was washed with PBST for 3 times, 10 min per time. The membranes were then incubated with secondary antibodies conjugated to horseradish peroxidase (ZSGB-BIO, ZB-2301, Beijing, China; ZSGB-BIO, ZB-2305, Beijing, China) at a dilution of 1:5000 and detected by the ECL System (ThermoFisher Scientific, SuperSignal™ West Femto Maximum Sensitivity Substrate, 34096, Massachusetts, USA).
Tissue collection and hematoxylin–eosin staining
Six Control or Ccnb3 mutant adult mouse testes and epididymis were collected and the testes were weighed, then fixed in 4% paraformaldehyde (PFA) for 48 h in 4 °C. The tissues were then embedded in paraffin. Five sections of each testis and epididymis (5 μm, taken 200 μm apart) were stained with hematoxylin–eosin (H&E) for normal histological analysis.
To test the fertility of the male mice, six 8-week-old control and six 8-week-old mutant mice were housed with wild-type proven fertility female C57 mice in a ratio of 1:2. Successful conception was defined by the presence of vaginal plug and subsequent visibly growing abdomen. The pregnant females were then separated and the litter sizes were recorded after birth.
All experiments were performed at least in triplicate and the results were presented as mean ± SEM. Two groups were compared by using Student’s t-test and P < 0.05 (*) were considered significant.