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Pogo transposable element derived with ZNF domain ( POGZ ) has been identified as one of the most recurrently de novo mutated genes in patients with neurodevelopmental disorders (NDDs), including autism spectrum disorder (ASD), intellectual disability and White-Sutton syndrome; however, the neurobiological basis behind these disorders remains unknown. Here, we show that POGZ regulates neuronal development and that ASD-related de novo mutations impair neuronal development in the developing mouse brain and induced pluripotent cell lines from an ASD patient. We also develop the first mouse model heterozygous for a de novo POGZ mutation identified in a patient with ASD, and we identify ASD-like abnormalities in the mice. Importantly, social deficits can be treated by compensatory inhibition of elevated cell excitability in the mice. Our results provide insight into how de novo mutations on high-confidence ASD genes lead to impaired mature cortical network function, which underlies the cellular pathogenesis of NDDs, including ASD. De novo mutations significantly contribute to autism spectrum disorders (ASD). Here, the authors demonstrate that ASD-associated de novo mutations in the POGZ gene, one of a high-confidence ASD gene, lead to ASD-related impaired neuronal development and disrupted mature cortical network function.

Pulmonary fibrosis is a progressive and fatal lung disease with limited therapeutic options. Although it is well known that lipid mediator prostaglandins are involved in the development of pulmonary fibrosis, the role of prostaglandin D2 (PGD2) remains unknown. Here, we investigated whether genetic disruption of hematopoietic PGD synthase (H-PGDS) affects the bleomycin-induced lung inflammation and pulmonary fibrosis in mouse. Compared with H-PGDS naïve (WT) mice, H-PGDS-deficient mice (H-PGDS-/-) represented increased collagen deposition in lungs 14 days after the bleomycin injection. The enhanced fibrotic response was accompanied by an increased mRNA expression of inflammatory mediators, including tumor necrosis factor-α, monocyte chemoattractant protein-1, and cyclooxygenase-2 on day 3. H-PGDS deficiency also increased vascular permeability on day 3 and infiltration of neutrophils and macrophages in lungs on day 3 and 7. Immunostaining showed that the neutrophils and macrophages expressed H-PGDS, and its mRNA expression was increased on day 3and 7 in WT lungs. These observations suggest that H-PGDS-derived PGD2 plays a protective role in bleomycin-induced lung inflammation and pulmonary fibrosis.

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

The RIKEN BioResource Research Center (BRC) was established in 2001 as a comprehensive biological resource center in Japan. The Experimental Animal Division, one of the BRC infrastructure divisions, has been designated as the core facility for mouse resources within the National BioResource Project (NBRP) by the Japanese government since FY2002. Our activities regarding the collection, preservation, quality control, and distribution of mouse resources have been supported by the research community, including evaluations and guidance on advancing social and research needs, as well as the operations and future direction of the BRC. Expenditure for collection, preservation, and quality-control operations of the BRC, as a national core facility, has been funded by the government, while distribution has been separately funded by users’ reimbursement fees. We have collected over 9000 strains created mainly by Japanese scientists including Nobel laureates and researchers in cutting-edge fields and distributed mice to 7000 scientists with 1500 organizations in Japan and globally. Our users have published 1000 outstanding papers and a few dozen patents. The collected mouse resources are accessible via the RIKEN BRC website, with a revised version of the searchable online catalog. In addition, to enhance the visibility of useful strains, we have launched web corners designated as the “Mouse of the Month” and “Today’s Tool and Model.” Only high-demand strains are maintained in live colonies, while other strains are cryopreserved as embryos or sperm to achieve cost-effective management. Since 2007, the RIKEN BRC has built up a back-up facility in the RIKEN Harima branch to protect the deposited strains from disasters. Our mice have been distributed with high quality through the application of strict microbial and genetic quality control programs that cover a globally accepted pathogens list and mutated alleles generated by various methods. Added value features, such as information about users’ publications, standardized phenotyping data, and genome sequences of the collected strains, are important to facilitate the use of our resources. We have added and disseminated such information in collaboration with the NBRP Information Center and the NBRP Genome Information Upgrading Program. The RIKEN BRC has participated in international mouse resource networks such as the International Mouse Strain Resource, International Mouse Phenotyping Consortium, and Asian Mouse Mutagenesis and Resource Association to facilitate the worldwide use of high-quality mouse resources, and as a consequence it contributes to reproducible life science studies and innovation around the globe.

A limited number of female germ cells support reproduction in many mammals. The follicle, composed of oocytes and supporting granulosa cells, forms the basis of oogenesis. Crosstalk between oocytes and granulosa cells is essential for the formation, dormancy, re-awakening, and maturation of oocytes. The oocyte expresses c-KIT and growth differentiation factor-9 (GDF-9), which are major factors in this crosstalk. The downstream signalling pathways of c-KIT and GDF-9 have been well-documented; however, their intra-oocyte trafficking pathway remains unclear. Our study reveals that the exocyst complex, a heterotetrameric protein complex important for tethering in vesicular transport, is important for proper intra-oocyte trafficking of c-KIT and GDF9 in mice. We found that depletion of oocyte-specific EXOC1, a component of the exocyst complex, impaired oocyte re-awakening and cyst breakdown, and inhibited granulosa cell proliferation during follicle growth. The c-KIT receptor is localised on the oocyte plasma membrane. The oocyte-specific Kit conditional knockout mice were reported to exhibit impaired oocyte re-awakening and reduced oocyte cyst breakdown. GDF9 is a protein secreted extracellularly in the oocyte. Previous studies have shown that Gdf9 knockout mice impaired proliferation and granulosa cell multilayering in growing follicles. We found that both c-KIT and GDF9 abnormally stuck in the EXOC1-depleted oocyte cytoplasm. These abnormal phenotypes were also observed in oocytes depleted of exocyst complex members EXOC3 and EXOC7. These results clearly show that the exocyst complex is essential for proper intra-oocyte trafficking of c-KIT and GDF9. Inhibition of this complex causes complete loss of female fertility in mice. Our findings build a platform for research related to trafficking mechanisms of vital crosstalk factors for oogenesis.

F1 hybrid progenies between related subspecies often show hybrid sterility (HS) or inviability. HS is caused by failure of meiotic chromosome synapsis and sex body formation in house mouse. Previous studies identified two HS critical genomic regions named Hstx2 on Chr X and Hst 1 on Chr 17 by murine forward genetic approaches. HS gene on Hst1 was reported to be Prdm9 . Intersubspecific polymorphisms of Prdm9 induce HS in hybrids, and Prdm9 null mutation leads to sterility in the inbred strain. However, HS gene on Hstx 2 remains unknown. Here, using knock-out studies, we showed that HS candidate genes on Hstx2 are not individually essential for spermatogenesis in B6 strain. We examined 12 genes on Hstx2 : Ctag2 , 49 3 0 4 47F04Rik , Mir743 , Mir46 5 d , Mir4 6 5c-2 , Mir465b-1 , Mir465c-1 , Mir465 , Gm1140 , Gm14692 , 4933436I01Rik , and Gm6812 . These genes were expressed in adult testes, and showed intersubspecific polymorphisms on expressed regions. This first reverse genetic approach to identify HS gene on Hstx2 suggested that the loss of function of any one HS candidate gene does not cause complete sterility, unlike Prdm9 . Thus, the mechanism(s) of HS by the HS gene on Hstx2 might be different from that of Prdm9 .

Genetically engineered mouse models are essential tools for understanding mammalian gene functions and disease pathogenesis. Genome editing allows the generation of these models in multiple inbred strains of mice without backcrossing. Zygote electroporation dramatically removed the barrier for introducing the CRISPR-Cas9 complex in terms of cost and labour. Here, we demonstrate that the generalised zygote electroporation method is also effective for generating knockout mice in multiple inbred strains. By combining in vitro fertilisation and electroporation, we obtained founders for knockout alleles in eight common inbred strains. Long-read sequencing analysis detected not only intended mutant alleles but also differences in read frequency of intended and unintended alleles among strains. Successful germline transmission of knockout alleles demonstrated that our approach can establish mutant mice targeting the same locus in multiple inbred strains for phenotyping analysis, contributing to reverse genetics and human disease research.

Purpose Penile research is expected to reveal new targets for treatment and prevention of the complex mechanisms of its disorder including erectile dysfunction (ED). Thus, analyses of the molecular processes of penile ED and continuous erection as priapism are essential issues of reproductive medicine. Methods By performing mouse N‐ethyl‐N‐nitrosourea mutagenesis and exome sequencing, we established a novel mouse line displaying protruded genitalia phenotype (PGP; priapism‐like phenotype) and identified a novel Pitpna gene mutation for PGP. Extensive histological analyses on the Pitpna mutant and intracavernous pressure measurement (ICP) and liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI/MS)/MS analyses were performed. Results We evaluated the role of phospholipids during erection for the first time and showed the mutants of inducible phenotypes of priapism. Moreover, quantitative analysis using LC–ESI/MS/MS revealed that the level of phosphatidylinositol (PI) was significantly lower in the mutant penile samples. These results imply that PI may contribute to penile erection by PITPα. Conclusions Our findings suggest that the current mutant is a mouse model for priapism and abnormalities in PI signaling pathways through PITPα may lead to priapism providing an attractive novel therapeutic target in its treatment.

The International Mouse Phenotyping Consortium reports the generation of new mouse mutant strains for more than 5,000 genes, including 2,850 novel null, 2,987 novel conditional-ready and 4,433 novel reporter alleles.

Background CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as “two-donor floxing” method). Results We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. Conclusion We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis , an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.