Mg influx mediated by TRPM7 triggers the initiation of muscle stem cell activation.

Muscle satellite cells (MuSCs) respond immediately to environmental cues upon skeletal muscle injuries. Despite decades of research into muscle regeneration, the specific molecular factors that trigger the transition of MuSCs from a quiescent to an active state remain largely unidentified. Here, we identify transient receptor potential melastatin 7 (TRPM7), an Mg-permeable ion channel, as a critical regulator of MuSC activation.

Pathological characterization of female reproductive organs prior to miscarriage induced by Zika virus infection in the pregnant common marmoset.

Unlabelled: While Zika virus (ZIKV) infection in pregnant women is known to increase the risk of miscarriage and stillbirth, the mechanism by which ZIKV infection leads to the inability to continue a pregnancy is not clear. In our common marmoset models of ZIKV infection in pregnant individuals, miscarriage was observed in dams infected in the first or second trimester, and preterm delivery was observed in a dam infected in the third trimester. Serum progesterone levels were significantly lower prior to miscarriage or preterm delivery in the infected marmosets.

Role of hepcidin upregulation and proteolytic cleavage of ferroportin 1 in hepatitis C virus-induced iron accumulation.

Hepatitis C virus (HCV) is a pathogen characterized not only by its persistent infection leading to the development of cirrhosis and hepatocellular carcinoma (HCC), but also by metabolic disorders such as lipid and iron dysregulation. Elevated iron load is commonly observed in the livers of patients with chronic hepatitis C, and hepatic iron overload is a highly profibrogenic and carcinogenic factor that increases the risk of HCC. However, the underlying mechanisms of elevated iron accumulation in HCV-infected livers remain to be fully elucidated.

The Mc4r gene is responsible for the development of experimentally induced testicular teratomas.

Teratomas in mice, composed of different tissue types, are derived from primordial germ cells in the fetal gonads. Previously, we identified a locus responsible for experimental testicular teratoma (ETT) formation on chromosome 18, referred to as ett1. The strongest candidate sequence in the ett1 locus was found to be a missense mutation in the melanocortin 4 receptor (Mc4r), Mc4r.

Generation of Flag/DYKDDDDK Epitope Tag Knock-In Mice Using -GONAD Enables Detection of Endogenous CaMKIIα and β Proteins.

Specific antibodies are necessary for cellular and tissue expression, biochemical, and functional analyses of protein complexes. However, generating a specific antibody is often time-consuming and effort-intensive. The epitope tagging of an endogenous protein at an appropriate position can overcome this problem.

Successful -GONAD in Mice at Early Zygote Stage through In Vivo Electroporation Three Min after Intraoviductal Instillation of CRISPR-Ribonucleoprotein.

Improved genome editing via oviductal nucleic acids delivery (-GONAD) is a new technology enabling in situ genome editing of mammalian zygotes exiting the oviductal lumen, which is now available in mice, rats, and hamsters. In this method, CRISPR/Cas9 genome-editing reagents are delivered directly to the oviducts of pregnant animals (corresponding to late zygote stage). After intraoviductal instillation, electric shock to the entire oviduct was provided with a specialized electroporation (EP) device to drive the genome editing reagents into the zygotes present in the oviductal lumen.

Recent Advances in the Production of Genome-Edited Rats.

The rat is an important animal model for understanding gene function and developing human disease models. Knocking out a gene function in rats was difficult until recently, when a series of genome editing (GE) technologies, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the type II bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated Cas9 (CRISPR/Cas9) systems were successfully applied for gene modification (as exemplified by gene-specific knockout and knock-in) in the endogenous target genes of various organisms including rats. Owing to its simple application for gene modification and its ease of use, the CRISPR/Cas9 system is now commonly used worldwide.

Modification of improved-genome editing via oviductal nucleic acids delivery (i-GONAD)-mediated knock-in in rats.

Background: Improved genome-editing via oviductal nucleic acids delivery (i-GONAD) is a new technology that facilitates in situ genome-editing of mammalian zygotes exiting the oviductal lumen. The i-GONAD technology has been developed for use in mice, rats, and hamsters; however, oligonucleotide (ODN)-based knock-in (KI) is more inefficient in rats than mice. To improve the efficiency of i-GONAD in rats we examined KI efficiency using three guide RNAs (gRNA), crRNA1, crRNA2 and crRNA3.

MC4R mutant mice develop ovarian teratomas.

Teratomas in mice, composed of different tissue types, are derived from primordial germ cells (PGCs) in the foetal gonads. The strongest candidate gene in the testicular teratoma locus (Ter) responsible for testicular teratoma formation was identified as mutation in Dnd1, Dnd1R178*. However, the phenotype of mice with a mutated Dnd1 gene was germ cell loss.

Modification of -GONAD Suitable for Production of Genome-Edited C57BL/6 Inbred Mouse Strain.

Improved genome editing via oviductal nucleic acid delivery (-GONAD) is a novel method for producing genome-edited mice in the absence of ex vivo handling of zygotes. -GONAD involves the intraoviductal injection of clustered regularly interspaced short palindromic repeats (CRISPR) ribonucleoproteins via the oviductal wall of pregnant females at 0.7 days post-coitum, followed by in vivo electroporation (EP).

Recent Advances and Future Perspectives of In Vivo Targeted Delivery of Genome-Editing Reagents to Germ Cells, Embryos, and Fetuses in Mice.

The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) systems that occur in nature as microbial adaptive immune systems are considered an important tool in assessing the function of genes of interest in various biological systems. Thus, development of efficient and simple methods to produce genome-edited (GE) animals would accelerate research in this field. The CRISPR/Cas9 system was initially employed in early embryos, utilizing classical gene delivery methods such as microinjection or electroporation, which required ex vivo handling of zygotes before transfer to recipients.

Sequential i-GONAD: An Improved In Vivo Technique for CRISPR/Cas9-Based Genetic Manipulations in Mice.

Improved genome-editing via oviductal nucleic acid delivery (i-GONAD) is a technique capable of inducing genomic changes in preimplantation embryos (zygotes) present within the oviduct of a pregnant female. i-GONAD involves intraoviductal injection of a solution containing genome-editing components via a glass micropipette under a dissecting microscope, followed by in vivo electroporation using tweezer-type electrodes. i-GONAD does not involve ex vivo handling of embryos (isolation of zygotes, microinjection or electroporation of zygotes, and egg transfer of the treated embryos to the oviducts of a recipient female), which is required for in vitro genome-editing of zygotes.

Nanopore sequencing reveals a structural alteration of mirror-image duplicated genes in a genome-editing mouse line.

CRISPR-Cas9 technology has been used in various studies; however, it has also been found to introduce unexpected structural alternations. In this study, we used nanopore sequencing to characterize an unexpected structural alteration of mirror-image duplicated genes in a mouse line, in which we aimed to delete a part of the duplicated genes using genome editing. We removed low-molecular-weight DNA fragments and increased the input, which led to improved sequence performance.

Creation of CRISPR-based germline-genome-engineered mice without ex vivo handling of zygotes by i-GONAD.

Methods to create genetically engineered mice involve three major steps: harvesting embryos from one set of females, microinjection of reagents into embryos ex vivo and their surgical transfer to another set of females. Although tedious, these methods have been used for more than three decades to create mouse models. We recently developed a method named GONAD (genome editing via oviductal nucleic acids delivery), which bypasses these steps.

i-GONAD (improved genome-editing via oviductal nucleic acids delivery), a convenient in vivo tool to produce genome-edited rats.

Zygote-microinjection or in vitro electroporation of isolated zygotes are now widely used methods to produce genome-edited mice. However, these technologies require laborious and time-consuming ex vivo handling of fertilized eggs, including zygote isolation, gene delivery into zygotes and embryo transfer into recipients. We recently developed an alternative method called improved genome-editing via oviductal nucleic acids delivery (i-GONAD), which does not require the above-mentioned ex vivo handing of zygotes, but instead involves intraoviductal instillation of genome-editing components, Cas9 protein and synthetic gRNAs, into the oviducts of pregnant females at the late 1-cell embryo stage under a dissecting microscope and subsequent electroporation.

Identification of Two Additional Genomic Loci Responsible for experimentally induced testicular teratoma 2 and 3 (ett2 and ett3).

Experimental testicular teratomas (ETTs) can be induced in 129/Sv mouse by E12.5 fetal testes transplant into adult testes. Previously, we conducted linkage analysis to explore candidate genes possibly involved in ETT development using F2 intercross fetuses derived from F1[LTXBJ × 129/Sv- + /Ter (+ /+)] hybrids.

i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases.

We present a robust method called improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) that delivers CRISPR ribonucleoproteins to E0.7 embryos via in situ electroporation. The method generates mouse models containing single-base changes, kilobase-sized deletions, and knock-ins.

The response of common marmoset immunity against cedar pollen extract.

The in vivo model of pollinosis has been established using rodents, but the model cannot completely mimic human pollinosis. We used Callithrix jacchus, the common marmoset (CM), to establish a pollinosis animal model using intranasal weekly administration of cedar pollen extract with cholera toxin adjuvant. Some of the treated CMs exhibited the symptoms of snitching, excess nasal mucus and/or sneezing, but the period was very short, and the symptoms disappeared after several weeks.

Critical role of CREBH-mediated induction of transforming growth factor β2 by hepatitis C virus infection in fibrogenic responses in hepatic stellate cells.

Unlabelled: Mechanisms of hepatic fibrogenesis induced by hepatitis C virus (HCV), one of the leading causes of liver fibrosis, are not fully understood. We studied transcriptional up-regulation of transforming growth factor β (TGF-β), especially TGF-β2, which is mediated by activation of liver-enriched transcription factor cAMP-responsive element-binding protein, hepatocyte specific (CREBH) triggered by HCV infection and its functional significance for induction of profibrogenic phenotypes by interaction of HCV-infected cells with hepatic stellate cells (HSCs). Compared to TGF-β1, expression of TGF-β2 mRNA was induced faster and to a higher level upon HCV infection.

Common marmoset CD117+ hematopoietic cells possess multipotency.

Analysis of the hematopoiesis of non-human primates is important to clarify the evolution of primate-specific hematopoiesis and immune regulation. However, the engraftment and development of the primate hematopoietic system are well-documented only in humans and are not clear in non-human primates. Callithrix jacchus (common marmoset, CM) is a New World monkey with a high rate of pregnancy and small size that lives in closed colonies.

Noninvasive genotyping of common marmoset (Callithrix jacchus) by fingernail PCR.

The common marmoset (Callithrix jacchus) is a New World primate that is a useful model for medical studies. In this study, we report a convenient, reliable, and noninvasive procedure to genotype a living common marmoset by using fingernails. This method was used to successfully genotype DNA by restriction fragment length polymorphism (RFLP) PCR without prior purification, by using the KOD FX PCR enzyme kit.

Development of a modified artificial insemination technique combining penile vibration stimulation and the swim-up method in the common marmoset.

The common marmoset, Callithrix jacchus, is used as a New World monkey species in biomedical studies because of its small body size and good reproduction in captivity. A modified artificial insemination technique was developed in this species to encourage breeding of lines carrying interesting genes and traits. Fresh semen was collected by penile vibratory stimulation.

Identification of genomic locus responsible for experimentally induced testicular teratoma 1 (ett1) on mouse Chr 18.

Spontaneous testicular teratomas (STTs) composed by various kinds of tissues are derived from primordial germ cells (PGCs) in the fetal testes of the mouse. In contrast, intra-testicular grafts of the mouse strain (129/Sv-Ter (+/+)) fetal testes possessed the ability to develop the experimental testicular teratomas (ETTs), indistinguishable from the STTs at a morphological level. In this study, linkage analysis was performed for exploration of possible candidate genes involving in ETT development using F2 intercross fetuses derived from [LTXBJ × 129/Sv-Ter (+/+)] F1 hybrids.

A new Enpp1 allele, Enpp1(ttw-Ham), identified in an ICR closed colony.

We recently have reported on a novel ankylosis gene that is closely linked to the Enpp1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) gene on chromosome 10. Here, we have discovered novel mutant mice in a Jcl:ICR closed colony with ankylosis in the toes of the forelimbs at about 3 weeks of age. The mutant mice exhibited rigidity in almost all joints, including the vertebral column, which increased with age.