Publications by authors named "Atsushi Yoshiki"

63 Publications

Quick validation of genetic quality for conditional alleles in mice.

Genes Cells 2021 Apr 26;26(4):240-245. Epub 2021 Feb 26.

Experimental Animal Division, RIKEN BioResource Research Center, Tsukuba, Japan.

Site-specific conditional inactivation technologies using Cre-loxP or Flp-FRT systems are becoming increasingly important for the elucidation of gene function and disease mechanism in vivo. A large number of gene knockout mouse models carrying complex conditional alleles have been generated by global community efforts and made available for biomedical researchers. The structures of conditional alleles in these mice are becoming increasingly complex and sophisticated, and so the validation of the genetic quality of these alleles is likewise becoming a laborious task for individual researchers. To ensure the reproducibility of conditional experiments, the researcher should confirm that loxP or FRT is integrated at the correct positions in the genome prior to start of the experiments. We report the successful design of universal PCR primers specific to loxP and FRT for the quick validation of conditional floxed and Flrted alleles. The primer set consists of forward and reverse primers complimentary to the loxP or FRT sequences with partial modifications at the 5' end containing 6-base restriction endonuclease recognition sites. The universal primer set was tested to detect genomic intervals between a pair of cis-integrated loxP or FRT and was useful for quickly validating various floxed or Flrted alleles in conditional mice.
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http://dx.doi.org/10.1111/gtc.12834DOI Listing
April 2021

Dynamic erectile responses of a novel penile organ model utilizing TPEM†.

Biol Reprod 2021 Apr;104(4):875-886

Department of Developmental Genetics, Institute of Advanced Medicine, Wakayama Medical University (WMU), Wakayama, Japan.

Male penis is required to become erect during copulation. In the upper (dorsal) part of penis, the erectile tissue termed corpus cavernosum (CC) plays fundamental roles for erection by regulating the inner blood flow. When blood flows into the CC, the microvascular complex termed sinusoidal space is reported to expand during erection. A novel in vitro explant system to analyze the dynamic erectile responses during contraction/relaxation is established. The current data show regulatory contraction/relaxation processes induced by phenylephrine (PE) and nitric oxide (NO) donor mimicking dynamic erectile responses by in vitro CC explants. Two-photon excitation microscopy (TPEM) observation shows the synchronous movement of sinusoidal space and the entire CC. By taking advantages of the CC explant system, tadalafil (Cialis) was shown to increase sinusoidal relaxation. Histopathological changes have been generally reported associating with erection in several pathological conditions. Various stressed statuses have been suggested to occur in the erectile responses by previous studies. The current CC explant model enables to analyze such conditions through directly manipulating CC in the repeated contraction/relaxation processes. Expression of oxidative stress marker and contraction-related genes, Hypoxia-inducible factor 1-alpha (Hif1a), glutathione peroxidase 1 (Gpx1), Ras homolog family member A (RhoA), and Rho-associated protein kinase (Rock), was significantly increased in such repeated contraction/relaxation. Altogether, it is suggested that the system is valuable for analyzing structural changes and physiological responses to several regulators in the field of penile medicine.
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http://dx.doi.org/10.1093/biolre/ioab011DOI Listing
April 2021

Establishment and application of information resource of mutant mice in RIKEN BioResource Research Center.

Lab Anim Res 2021 Jan 18;37(1). Epub 2021 Jan 18.

Integrated Bioresource Information Division, RIKEN BioResource Research Center, 3-1-1 Koyadai, Tsukuba-shi, Ibaraki, 305-0074, Japan.

Online databases are crucial infrastructures to facilitate the wide effective and efficient use of mouse mutant resources in life sciences. The number and types of mouse resources have been rapidly growing due to the development of genetic modification technology with associated information of genomic sequence and phenotypes. Therefore, data integration technologies to improve the findability, accessibility, interoperability, and reusability of mouse strain data becomes essential for mouse strain repositories. In 2020, the RIKEN BioResource Research Center released an integrated database of bioresources including, experimental mouse strains, Arabidopsis thaliana as a laboratory plant, cell lines, microorganisms, and genetic materials using Resource Description Framework-related technologies. The integrated database shows multiple advanced features for the dissemination of bioresource information. The current version of our online catalog of mouse strains which functions as a part of the integrated database of bioresources is available from search bars on the page of the Center ( https://brc.riken.jp ) and the Experimental Animal Division ( https://mus.brc.riken.jp/ ) websites. The BioResource Research Center also released a genomic variation database of mouse strains established in Japan and Western Europe, MoG ( https://molossinus.brc.riken.jp/mogplus/ ), and a database for phenotype-phenotype associations across the mouse phenome using data from the International Mouse Phenotyping Platform. In this review, we describe features of current version of databases related to mouse strain resources in RIKEN BioResource Research Center and discuss future views.
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http://dx.doi.org/10.1186/s42826-020-00068-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7811887PMC
January 2021

Substrains matter in phenotyping of C57BL/6 mice.

Exp Anim 2021 May 14;70(2):145-160. Epub 2021 Jan 14.

Experimental Animal Division, RIKEN BioResource Research Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan.

The inbred mouse strain C57BL/6 has been widely used as a background strain for spontaneous and induced mutations. Developed in the 1930s, the C57BL/6 strain diverged into two major groups in the 1950s, namely, C57BL/6J and C57BL/6N, and more than 20 substrains have been established from them worldwide. We previously reported genetic differences among C57BL/6 substrains in 2009 and 2015. Since then, dozens of reports have been published on phenotypic differences in behavioral, neurological, cardiovascular, and metabolic traits. Substrains need to be chosen according to the purpose of the study because phenotypic differences might affect the experimental results. In this paper, we review recent reports of phenotypic and genetic differences among C57BL/6 substrains, focus our attention on the proper use of C57BL/6 and other inbred strains in the era of genome editing, and provide the life science research community wider knowledge about this subject.
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http://dx.doi.org/10.1538/expanim.20-0158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8150240PMC
May 2021

Development of assisted reproductive technologies for Mus spretus†.

Biol Reprod 2021 01;104(1):234-243

RIKEN BioResouce Research Center, Tsukuba, Japan.

The genus Mus consists of many species with high genetic diversity. However, only one species, Mus musculus (the laboratory mouse), is common in biomedical research. The unavailability of assisted reproductive technologies (ARTs) for other Mus species might be a major reason for their limited use in laboratories. Here, we devised ARTs for Mus spretus (the Algerian mouse), a commonly used wild-derived Mus species. We found that in vitro production of M. spretus embryos was difficult because of low efficacies of superovulation with equine chorionic gonadotropin or anti-inhibin serum (AIS) (5-8 oocytes per female) and a low fertilization rate following in vitro fertilization (IVF; 15.2%). The primary cause of this was the hardening of the zona pellucida but not the sperm's fertilizing ability, as revealed by reciprocal IVF with laboratory mice. The largest number of embryos (16 per female) were obtained when females were injected with AIS followed by human chorionic gonadotropin and estradiol injections 24 h later, and then by natural mating. These in vivo-derived 2-cell embryos could be vitrified/warmed with a high survival rate (94%) using an ethylene glycol-based solution. Importantly, more than 60% of such embryos developed into healthy offspring following interspecific embryo transfer into (C57BL/6 × C3H) F1 female mice. Thus, we have devised practical ARTs for Mus spretus mice, enabling efficient production of embryos and animals, with safe laboratory preservation of their strains. In addition, we have demonstrated that interspecific embryo transfer is possible in murine rodents.
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http://dx.doi.org/10.1093/biolre/ioaa177DOI Listing
January 2021

Characterization of a bicistronic knock-in reporter mouse model for investigating the role of CABLES2 in vivo.

Exp Anim 2021 Feb 11;70(1):22-30. Epub 2020 Aug 11.

Laboratory Animal Resource Center, Trans-Border Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.

Two members of the CDK5 and ABL enzyme substrate (CABLES) family, CABLES1 and CABLES2, share a highly homologous C-terminus. They interact and associate with cyclin-dependent kinase 3 (CDK3), CDK5, and c-ABL. CABLES1 mediates tumor suppression, regulates cell proliferation, and prevents protein degradation. Although Cables2 is ubiquitously expressed in adult mouse tissues at RNA level, the role of CABLES2 in vivo remains unknown. Here, we generated bicistronic Cables2 knock-in reporter mice that expressed CABLES2 tagged with 3×FLAG and 2A-mediated fluorescent reporter tdTomato. Cables2-3×FLAG-2A-tdTomato (Cables2) mice confirmed the expression of Cables2 in various mouse tissues. Interestingly, high intensity of tdTomato fluorescence was observed in the brain, testis and ovary, especially in the corpus luteum. Furthermore, immunoprecipitation analysis using the brain and testis in Cables2 revealed interaction of CABLES2 with CDK5. Collectively, our new Cables2 knock-in reporter model will enable the comprehensive analysis of in vivo CABLES2 function.
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http://dx.doi.org/10.1538/expanim.20-0063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7887623PMC
February 2021

Reverse genetics reveals single gene of every candidate on Hybrid sterility, X Chromosome QTL 2 (Hstx2) are dispensable for spermatogenesis.

Sci Rep 2020 06 3;10(1):9060. Epub 2020 Jun 3.

Laboratory Animal Resource Center and Trans-border Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8575, Japan.

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 Hst1 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 Hstx2 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, 4930447F04Rik, Mir743, Mir465d, Mir465c-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.
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http://dx.doi.org/10.1038/s41598-020-65986-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7270182PMC
June 2020

Diverse dystonin gene mutations cause distinct patterns of isoform deficiency and phenotypic heterogeneity in mice.

Dis Model Mech 2020 05 21;13(5). Epub 2020 May 21.

Division of Neurobiology and Anatomy, Graduate School of Medical and Dental Sciences, Niigata University, Niigata 951-8510, Japan

Loss-of-function mutations in dystonin () can cause hereditary sensory and autonomic neuropathy type 6 (HSAN-VI) or epidermolysis bullosa simplex (EBS). Recently, -related diseases were recognized to be more complex than previously thought because a patient exhibited both neurological and skin manifestations, whereas others display only one or the other. A single locus produces at least three major isoforms: (neuronal isoform), (muscular isoform) and (epithelial isoform). () mice, which have mutations in , were originally identified as spontaneous mutants displaying neurological phenotypes. To reveal the mechanisms underlying the phenotypic heterogeneity of -related diseases, we investigated two mutant strains with different mutations: a spontaneous mutant ( mice) and a gene-trap mutant ( mice). The allele possesses a nonsense mutation in an exon shared by all isoforms. The allele is predicted to inactivate and isoforms but not There was a decrease in the levels of mRNA in the neural tissue of both and homozygotes. Loss of sensory and autonomic nerve ends in the skin was observed in both and mice at postnatal stages. In contrast, mRNA expression was reduced in the skin of mice but not in mice. Expression levels of Dst proteins in neural and cutaneous tissues correlated with mRNAs. Because encodes a structural protein in hemidesmosomes (HDs), we performed transmission electron microscopy. Lack of inner plaques and loss of keratin filament invasions underneath the HDs were observed in the basal keratinocytes of mice but not in those of mice; thus, the distinct phenotype of the skin of mice could be because of failure of Dst-e expression. These results indicate that distinct mutations within the locus can cause different loss-of-function patterns among isoforms, which accounts for the heterogeneous neural and skin phenotypes in mice and -related diseases.
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http://dx.doi.org/10.1242/dmm.041608DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7325434PMC
May 2020

Efficient production of large deletion and gene fragment knock-in mice mediated by genome editing with Cas9-mouse Cdt1 in mouse zygotes.

Methods 2021 07 22;191:23-31. Epub 2020 Apr 22.

Laborarory Animal Resource Center, Transborder Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.

Genetically modified mouse models are essential for in vivo investigation of gene function and human disease research. Targeted mutations can be introduced into mouse embryos using genome editing technology such as CRISPR-Cas. Although mice with small indel mutations can be produced, the production of mice carrying large deletions or gene fragment knock-in alleles remains inefficient. We introduced the nuclear localisation property of Cdt1 protein into the CRISPR-Cas system for efficient production of genetically engineered mice. Mouse Cdt1-connected Cas9 (Cas9-mC) was present in the nucleus of HEK293T cells and mouse embryos. Cas9-mC induced a bi-allelic full deletion of Dmd, GC-rich fragment knock-in, and floxed allele knock-in with high efficiency compared to standard Cas9. These results indicate that Cas9-mC is a useful tool for producing mouse models carrying targeted mutations.
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http://dx.doi.org/10.1016/j.ymeth.2020.04.007DOI Listing
July 2021

Pathogenic POGZ mutation causes impaired cortical development and reversible autism-like phenotypes.

Nat Commun 2020 02 26;11(1):859. Epub 2020 Feb 26.

Technology and Developmental Team for Mouse Phenotype Analysis, RIKEN BioResource Research Center, Tsukuba, Ibaraki, 305-0074, Japan.

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.
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http://dx.doi.org/10.1038/s41467-020-14697-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7044294PMC
February 2020

Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation.

Genome Biol 2019 08 26;20(1):171. Epub 2019 Aug 26.

Department of Basic Medicine, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, 143, Shimokasuya, Isehara, Kanagawa, 259-1193, Japan.

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.
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http://dx.doi.org/10.1186/s13059-019-1776-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6709553PMC
August 2019

Off- and on-target effects of genome editing in mouse embryos.

J Reprod Dev 2019 Feb 6;65(1):1-5. Epub 2018 Dec 6.

Experimental Animal Division, RIKEN BioResource Research Center, Ibaraki 305-0074, Japan.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-based genome editing technology has enabled manipulation of the embryonic genome. Unbiased whole genome sequencing comparing parents to progeny has revealed that the rate of Cas9-induced mutagenesis in mouse embryos is indistinguishable from the background rate of de novo mutation. However, establishing the best practice to confirm on-target alleles of interest remains a challenge. We believe that improvement in editing strategies and screening methods for founder mice will contribute to the generation of quality-controlled animals, thereby ensuring reproducibility of results in animal studies and advancing the 3Rs (replacement, reduction, and refinement).
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http://dx.doi.org/10.1262/jrd.2018-128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379761PMC
February 2019

Macrophage-Associated Gelatinase Degrades Basement Membrane at the Optic Fissure Margins During Normal Ocular Development in Mice.

Invest Ophthalmol Vis Sci 2018 03;59(3):1368-1373

Laboratory of Pathology, Faculty of Pharmaceutical Sciences, Setsunan University, Osaka, Japan.

Purpose: Basement membrane degradation and macrophage aggregation at the optic fissure margins are crucial to optic fissure closure during normal murine eye development. Basement membrane degradation is also an essential step in cancer development, and matrix metalloproteinases (MMPs) play an important role. In this study, we investigated MMP alteration at the degrading basement membrane of optic fissure margins in mice and attempted to clarify the relationship between MMP activity and macrophages.

Methods: Serial coronal frozen sections of eyes from BALB/c fetuses were prepared and gelatinase activity was examined using in situ zymography techniques. The frozen sections were immunohistochemically stained with anti-F4/80, anti-MMP 2, and anti-MMP 9 antibodies. Serial coronal paraffin sections were also immunohistochemically stained with anti-type IV collagen and anti-F4/80, and basement membrane disintegration and macrophage aggregation at the optic fissure margins were examined.

Results: The basement membrane of optic fissure margins was rapidly degraded during gestational days (GDs) 12.0 to 12.5. Meanwhile, gelatinase activity at F4/80-positive macrophages significantly increased during GDs 11.5 to 12.0 and declined thereafter; some of those were also positive for MMP2. The number of macrophages was also increased and decreased at nearly the same time.

Conclusions: Intramacrophage MMPs may be responsible for basement membrane degradation at the optic fissure margins during normal eye development in mice.
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http://dx.doi.org/10.1167/iovs.17-21841DOI Listing
March 2018

Distinct Neural Circuits for the Formation and Retrieval of Episodic Memories.

Cell 2017 Aug 17;170(5):1000-1012.e19. Epub 2017 Aug 17.

RIKEN-MIT Center for Neural Circuit Genetics at the Picower Institute for Learning and Memory, Department of Biology and Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. Electronic address:

The formation and retrieval of a memory is thought to be accomplished by activation and reactivation, respectively, of the memory-holding cells (engram cells) by a common set of neural circuits, but this hypothesis has not been established. The medial temporal-lobe system is essential for the formation and retrieval of episodic memory for which individual hippocampal subfields and entorhinal cortex layers contribute by carrying out specific functions. One subfield whose function is poorly known is the subiculum. Here, we show that dorsal subiculum and the circuit, CA1 to dorsal subiculum to medial entorhinal cortex layer 5, play a crucial role selectively in the retrieval of episodic memories. Conversely, the direct CA1 to medial entorhinal cortex layer 5 circuit is essential specifically for memory formation. Our data suggest that the subiculum-containing detour loop is dedicated to meet the requirements associated with recall such as rapid memory updating and retrieval-driven instinctive fear responses.
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http://dx.doi.org/10.1016/j.cell.2017.07.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5586038PMC
August 2017

Characterization of novel dystonia musculorum mutant mice: Implications for central nervous system abnormality.

Neurobiol Dis 2016 Dec 28;96:271-283. Epub 2016 Sep 28.

Division of Neurobiology and Anatomy, Niigata University, Niigata 951-8510, Japan. Electronic address:

We identified a novel spontaneous mutant mouse showing motor symptoms that are similar to those of the dystonia musculorum (dt) mouse. The observations suggested that the mutant mice inherited the mild dt phenotype as an autosomal recessive trait. Linkage analysis showed that the causative gene was located near D1Mit373 and D1Mit410 microsatellite markers on chromosome 1, which are close to the dystonin (Dst) gene locus. To investigate whether Dst is the causative gene of the novel mutant phenotype, we crossed the mutant with Dst gene trap (Dst) mice. Compound heterozygotes showed a typical dt phenotype with sensory degeneration and progressive motor symptoms. DNA sequencing analysis identified a nonsense mutation within the spectrin repeats of the plakin domain. The novel mutant allele was named dt. Motor abnormalities in homozygous dt/dt mice are not as severe as homozygous Dst/Dst mice. Histological analyses showed abnormal neurofilament (NF) accumulation in the nervous system of homozygous dt/dt mice, which is characteristic of the dt phenotype. We mapped the distribution of abnormal NF-accumulated neurons in the brain and found that they were located specifically in the brainstem, spinal cord, and in regions such as the vestibular nucleus, reticular nucleus, and red nucleus, which are implicated in posture and motor coordination pathways. The quantification of abnormal NF accumulation in the cytoplasm and spheroids (axons) of neurons showed that abnormal NF immunoreactivity was lower in homozygous dt/dt mice than in homozygous Dst/Dst mice. Therefore, we have identified a novel hypomorphic allele of dt, which causes histological abnormalities in the central nervous system that may account for the abnormal motor phenotype. This novel spontaneously occurring mutant may become a good model of hereditary sensory and autonomic neuropathy type 6, which is caused by mutations in the human DST gene.
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http://dx.doi.org/10.1016/j.nbd.2016.09.016DOI Listing
December 2016

High-throughput discovery of novel developmental phenotypes.

Nature 2016 09 14;537(7621):508-514. Epub 2016 Sep 14.

Department of Molecular Physiology and Biophysics, Houston, Texas 77030, USA.

Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.
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http://dx.doi.org/10.1038/nature19356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5295821PMC
September 2016

Germline recombination in a novel Cre transgenic line, Prl3b1-Cre mouse.

Genesis 2016 07 13;54(7):389-97. Epub 2016 May 13.

Laboratory Animal Resource Center, University of Tsukuba, Tsukuba, Ibaraki, Japan.

Spermatogenesis is a complex and highly regulated process by which spermatogonial stem cells differentiate into spermatozoa. To better understand the molecular mechanisms of the process, the Cre/loxP system has been widely utilized for conditional gene knockout in mice. In this study, we generated a transgenic mouse line that expresses Cre recombinase under the control of the 2.5 kbp of the Prolactin family 3, subfamily b, member 1 (Prl3b1) gene promoter (Prl3b1-cre). Prl3b1 was initially reported to code for placental lactogen 2 (PL-2) protein in placenta along with increased expression toward the end of pregnancy. PL-2 was found to be expressed in germ cells in the testis, especially in spermatocytes. To analyze the specificity and efficiency of Cre recombinase activity in Prl3b1-cre mice, the mice were mated with reporter R26GRR mice, which express GFP ubiquitously before and tdsRed exclusively after Cre recombination. The systemic examination of Prl3b1-cre;R26GRR mice revealed that tdsRed-positive cells were detected only in the testis and epididymis. Fluorescence imaging of Prl3b1-cre;R26GRR testes suggested that Cre-mediated recombination took place in the germ cells with approximately 74% efficiency determined by in vitro fertilization. In conclusion, our results suggest that the Prl3b1-cre mice line provides a unique resource to understand testicular germ-cell development. genesis 54:389-397, 2016. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/dvg.22944DOI Listing
July 2016

Generation of CRISPR/Cas9-mediated bicistronic knock-in ins1-cre driver mice.

Exp Anim 2016 Jul 7;65(3):319-27. Epub 2016 Apr 7.

Laborarory Animal Resource Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.

In the present study, we generated novel cre driver mice for gene manipulation in pancreatic β cells. Using the CRISPR/Cas9 system, stop codon sequences of Ins1 were targeted for insertion of cre, including 2A sequences. A founder of C57BL/6J-Ins1(em1 (cre) Utr) strain was produced from an oocyte injected with pX330 containing the sequences encoding gRNA and Cas9 and a DNA donor plasmid carrying 2A-cre. (R26GRR x C57BL/6J-Ins1(em1 (cre) Utr)) F1 mice were histologically characterized for cre-loxP recombination in the embryonic and adult stages; cre-loxP recombination was observed in all pancreatic islets examined in which almost all insulin-positive cells showed tdsRed fluorescence, suggesting β cell-specific recombination. Furthermore, there were no significant differences in results of glucose tolerance test among genotypes (homo/hetero/wild). Taken together, these observations indicated that C57BL/6J-Ins1(em1 (cre) Utr) is useful for studies of glucose metabolism and the strategy of bicistronic cre knock-in using the CRISPR/Cas9 system could be useful for production of cre driver mice.
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http://dx.doi.org/10.1538/expanim.16-0016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4976246PMC
July 2016

NIG_MoG: a mouse genome navigator for exploring intersubspecific genetic polymorphisms.

Mamm Genome 2015 Aug 27;26(7-8):331-7. Epub 2015 May 27.

Mammalian Genetics Laboratory, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka, 411-8540, Japan,

The National Institute of Genetics Mouse Genome database (NIG_MoG; http://molossinus.lab.nig.ac.jp/msmdb/) primarily comprises the whole-genome sequence data of two inbred mouse strains, MSM/Ms and JF1/Ms. These strains were established at NIG and originated from the Japanese subspecies Mus musculus molossinus. NIG_MoG provides visualized genome polymorphism information, browsing single-nucleotide polymorphisms and short insertions and deletions in the genomes of MSM/Ms and JF1/Ms with respect to C57BL/6J (whose genome is predominantly derived from the West European subspecies M. m. domesticus). This allows users, especially wet-lab biologists, to intuitively recognize intersubspecific genome divergence in these mouse strains using visual data. The database also supports the in silico screening of bacterial artificial chromosome (BAC) clones that contain genomic DNA from MSM/Ms and the standard classical laboratory strain C57BL/6N. NIG_MoG is thus a valuable navigator for exploring mouse genome polymorphisms and BAC clones that are useful for studies of gene function and regulation based on intersubspecific genome divergence.
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http://dx.doi.org/10.1007/s00335-015-9569-8DOI Listing
August 2015

A simple and robust method for establishing homogeneous mouse epiblast stem cell lines by wnt inhibition.

Stem Cell Reports 2015 Apr 26;4(4):744-57. Epub 2015 Mar 26.

Technology and Development Team for Mammalian Genome Dynamics, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan; Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki 305-8572, Japan. Electronic address:

Epiblast stem cells (EpiSCs) are pluripotent stem cells derived from epiblasts of postimplantation mouse embryos, and thus provide a useful model for studying "primed" pluripotent states. Here, we devised a simple and robust technique to derive high-quality EpiSCs using an inhibitor of WNT secretion. Using this method, we readily established EpiSC lines with high efficiency and were able to use whole embryonic portions without having to separate the epiblast from the visceral endoderm (VE). Expression analyses revealed that these EpiSCs maintained a homogeneous, undifferentiated status, yet showed high potential for differentiation both in vitro and in teratomas. Unlike EpiSCs derived by the original protocol, new EpiSC lines required continuous treatment with the Wnt inhibitor, suggesting some intrinsic differences from the existing EpiSCs. The homogeneous properties of this new version of EpiSCs should facilitate studies on the establishment and maintenance of a "primed" pluripotent state, and directed differentiation from the primed state.
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http://dx.doi.org/10.1016/j.stemcr.2015.02.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4400648PMC
April 2015

Effects of background mutations and single nucleotide polymorphisms (SNPs) on the Disc1 L100P behavioral phenotype associated with schizophrenia in mice.

Behav Brain Funct 2014 Dec 8;10:45. Epub 2014 Dec 8.

Department of Biological Psychiatry and Neuroscience, Dokkyo Medical University School of Medicine, 800 Kitakobayashi, Mibu-machi, Shimotsuga-gun, Tochigi 321-0293, Japan.

Background: Disrupted-in-schizophrenia 1 (DISC1) is a promising candidate susceptibility gene for psychiatric disorders, including schizophrenia, bipolar disorder and major depression. Several previous studies reported that mice with N-ethyl-N-nitrosourea (ENU)-induced L100P mutation in Disc1 showed some schizophrenia-related behavioral phenotypes. This line originally carried several thousands of ENU-induced point mutations in the C57BL/6 J strain and single nucleotide polymorphisms (SNPs) from the DBA/2 J inbred strain.

Methods: To investigate the effect of Disc1 L100P, background mutations and SNPs on phenotypic characterization, we performed behavioral analyses to better understand phenotypes of Disc1 L100P mice and comprehensive genetic analyses using whole-exome resequencing and SNP panels to map ENU-induced mutations and strain-specific SNPs, respectively.

Results: We found no differences in spontaneous or methamphetamine-induced locomotor activity, sociability or social novelty preference among Disc1 L100P/L100P, L100P/+ mutants and wild-type littermates. Whole-exome resequencing of the original G1 mouse identified 117 ENU-induced variants, including Disc1 L100P per se. Two females and three males from the congenic L100P strain after backcrossing to C57BL/6 J were deposited to RIKEN BioResource Center in 2008. We genotyped them with DBA/2 J × C57BL/6 J SNPs and found a number of the checked SNPs still remained.

Conclusion: These results suggest that causal attribution of the discrepancy in behavioral phenotypes to the Disc1 L100P mutant mouse line existing among different research groups needs to be cautiously investigated in further study by taking into account the effect(s) of other ENU-induced mutations and/or SNPs from DBA/2 J.
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http://dx.doi.org/10.1186/1744-9081-10-45DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4295473PMC
December 2014

Devising assisted reproductive technologies for wild-derived strains of mice: 37 strains from five subspecies of Mus musculus.

PLoS One 2014 3;9(12):e114305. Epub 2014 Dec 3.

RIKEN BioResouce Center, Tsukuba, Ibaraki, Japan; Graduate School of Life and Environmental Science, University of Tsukuba, Tsukuba, Ibaraki, Japan; Center for Disease Biology and Integrative Medicine, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan.

Wild-derived mice have long offered invaluable experimental models for mouse genetics because of their high evolutionary divergence from laboratory mice. A number of wild-derived strains are available from the RIKEN BioResource Center (BRC), but they have been maintained as living stocks because of the unavailability of assisted reproductive technology (ART). In this study, we sought to devise ART for 37 wild-derived strains from five subspecies of Mus musculus maintained at the BRC. Superovulation of females was effective (more than 15 oocytes per female) for 34 out of 37 strains by treatment with either equine chorionic gonadotropin or anti-inhibin serum, depending on their genetic background (subspecies). The collected oocytes could be fertilized in vitro at mean rates of 79.0% and 54.6% by the optimized protocol using fresh or frozen-thawed spermatozoa, respectively. They were cryopreserved at the 2-cell stage by vitrification with an ethylene glycol-based solution. In total, 94.6% of cryopreserved embryos survived the vitrification procedure and restored their normal morphology after warming. A conventional embryo transfer protocol could be applied to 25 out of the 35 strains tested. In the remaining 10 strains, live offspring could be obtained by a modified embryo transfer protocol using cyclosporin A treatment and co-transfer of ICR (laboratory mouse strain) embryos. Thus, ART for 37 wild-derived strains was devised successfully and is now routinely used for their preservation and transportation. The information provided here might facilitate broader use and wider distribution of wild-derived mice for biomedical research.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0114305PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4254977PMC
December 2015

Development of SNP markers for C57BL/6N-derived mouse inbred strains.

Exp Anim 2015 23;64(1):91-100. Epub 2014 Oct 23.

Experimental Animal Division, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan.

C57BL/6N inbred mice are used as the genetic background for producing knockout mice in large-scale projects worldwide; however, the genetic divergence among C57BL/6N-derived substrains has not been verified. Here, we identified novel single nucleotide polymorphisms (SNPs) specific to the C57BL/6NJ strain and selected useful SNPs for the genetic monitoring of C57BL/6N-derived substrains. Informative SNPs were selected from the public SNP database at the Wellcome Trust Sanger Institute by comparing sequence data from C57BL/6NJ and C57BL/6J mice. A total of 1,361 candidate SNPs from the SNP database could distinguish the C57BL/6NJ strain from 12 other inbred strains. We confirmed 277 C57BL/6NJ-specific SNPs including 10 nonsynonymous SNPs by direct sequencing, and selected 100 useful SNPs that cover all of the chromosomes except Y. Genotyping of 11 C57BL/6N-derived substrains at these 100 SNP loci demonstrated genetic differences among the substrains. This information will be useful for accurate genetic monitoring of mouse strains with a C57BL/6N-derived background.
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http://dx.doi.org/10.1538/expanim.14-0061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4329520PMC
September 2015

Generation and characterization of Ins1-cre-driver C57BL/6N for exclusive pancreatic beta cell-specific Cre-loxP recombination.

Exp Anim 2014 ;63(2):183-91

Laborarory Animal Resource Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575, Japan.

Cre/loxP system-mediated site-specific recombination is utilized to study gene function in vivo. Successful conditional knockout of genes of interest is dependent on the availability of Cre-driver mice. We produced and characterized pancreatic β cell-specific Cre-driver mice for use in diabetes mellitus research. The gene encoding Cre was inserted into the second exon of mouse Ins1 in a bacterial artificial chromosome (BAC). Five founder mice were produced by microinjection of linearized BAC Ins1-cre. The transgene was integrated between Mafa and the telomere on chromosome 15 in one of the founders, BAC Ins1-cre25. To investigate Cre-loxP recombination, BAC Ins1-cre25 males were crossed with two different Cre-reporters, R26R and R26GRR females. On gross observation, reporter signal after Cre-loxP recombination was detected exclusively in the adult pancreatic islets in both F1 mice. Immunohistological analysis indicated that Cre-loxP recombination-mediated reporter signal was colocalized with insulin in pancreatic islet cells of both F1 mice, but not with glucagon. Moreover, Cre-loxP recombination signal was already observed in the pancreatic islets at E13.5 in both F1 fetuses. Finally, we investigated ectopic Cre-loxP recombination for Ins1, because the ortholog Ins2 is also expressed in the brain, in addition to the pancreas. However, there was no Cre-loxP recombination-mediated reporter signal in the brain of both F1 mice. Our data suggest that BAC Ins1-cre25 mice are a useful Cre-driver C57BL/6N for pancreatic β cell-specific Cre-loxP recombination, except for crossing with knock-in mice carrying floxed gene on chromosome 15.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4160984PMC
http://dx.doi.org/10.1538/expanim.63.183DOI Listing
October 2014

Island cells control temporal association memory.

Science 2014 Feb 23;343(6173):896-901. Epub 2014 Jan 23.

RIKEN-MIT Center for Neural Circuit Genetics at the Picower Institute for Learning and Memory, Department of Biology and Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA.

Episodic memory requires associations of temporally discontiguous events. In the entorhinal-hippocampal network, temporal associations are driven by a direct pathway from layer III of the medial entorhinal cortex (MECIII) to the hippocampal CA1 region. However, the identification of neural circuits that regulate this association has remained unknown. In layer II of entorhinal cortex (ECII), we report clusters of excitatory neurons called island cells, which appear in a curvilinear matrix of bulblike structures, directly project to CA1, and activate interneurons that target the distal dendrites of CA1 pyramidal neurons. Island cells suppress the excitatory MECIII input through the feed-forward inhibition to control the strength and duration of temporal association in trace fear memory. Together, the two EC inputs compose a control circuit for temporal association memory.
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http://dx.doi.org/10.1126/science.1244634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5572219PMC
February 2014

Novel ROSA26 Cre-reporter knock-in C57BL/6N mice exhibiting green emission before and red emission after Cre-mediated recombination.

Exp Anim 2013 ;62(4):295-304

Laborarory Animal Resource Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575, Japan.

The Cre/loxP system is a strategy for controlling temporal and/or spatial gene expression through genome alteration in mice. As successful Cre/loxP genome alteration depends on Cre-driver mice, Cre-reporter mice are essential for validation of Cre gene expression in vivo. In most Cre-reporter mouse strains, although the presence of reporter product indicates the expression of Cre recombinase, it has remained unclear whether a lack of reporter signal indicates either no Cre recombinase expression or insufficient reporter gene promoter activity. We produced a novel ROSA26 knock-in Cre-reporter C57BL/6N strain exhibiting green emission before and red after Cre-mediated recombination, designated as strain R26GRR. Ubiquitous green fluorescence and no red fluorescence were observed in R26GRR mice. To investigate the activation of tdsRed, EGFP-excised R26GRR, R26RR, mice were produced through the crossing of C57BL/6N mice with R26GRR/Ayu1-Cre F1 mice. R26RR mice showed extraordinarily strong red fluorescence in almost all tissues examined, suggesting ubiquitous activation of the second reporter in all tissues after Cre/loxP recombination. Moreover, endothelial cell lineage and pancreatic islet-specific expression of red fluorescence were detected in R26GRR/Tie2-Cre F1 mice and R26GRR /Ins1-Cre F1 mice, respectively. These results indicated that R26GRR mice are a useful novel Cre-reporter mouse strain. In addition, R26GRR mice with a pure C57BL/6N background represent a valuable source of green-to-red photoconvertible cells following Cre/loxP recombination for application in transplantation studies. The R26GRR mouse strain will be available from RIKEN BioResource Center (http://www.brc.riken.jp/lab/animal/en/).
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4160954PMC
http://dx.doi.org/10.1538/expanim.62.295DOI Listing
April 2014

Understanding the X chromosome inactivation cycle in mice: a comprehensive view provided by nuclear transfer.

Epigenetics 2014 Feb 29;9(2):204-11. Epub 2013 Oct 29.

RIKEN BioResource Center; Tsukuba, Japan; Graduate School of Life and Environmental Science; University of Tsukuba; Tsukuba, Japan; Center for Disease Biology and Integrative Medicine; University of Tokyo; Tokyo, Japan.

During mouse development, imprinted X chromosome inactivation (XCI) is observed in preimplantation embryos and is inherited to the placental lineage, whereas random XCI is initiated in the embryonic proper. Xist RNA, which triggers XCI, is expressed ectopically in cloned embryos produced by somatic cell nuclear transfer (SCNT). To understand these mechanisms, we undertook a large-scale nuclear transfer study using different donor cells throughout the life cycle. The Xist expression patterns in the reconstructed embryos suggested that the nature of imprinted XCI is the maternal Xist-repressing imprint established at the last stage of oogenesis. Contrary to the prevailing model, this maternal imprint is erased in both the embryonic and extraembryonic lineages. The lack of the Xist-repressing imprint in the postimplantation somatic cells clearly explains how the SCNT embryos undergo ectopic Xist expression. Our data provide a comprehensive view of the XCI cycle in mice, which is essential information for future investigations of XCI mechanisms.
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http://dx.doi.org/10.4161/epi.26939DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962530PMC
February 2014