We present a robust method called improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) that delivers CRISPR ribonucleoproteins to E0.7 embryos in situ electroporation. The generates mouse models containing single-base changes, kilobase-sized deletions, and knock-ins. efficiency of i-GONAD is comparable traditional microinjection methods, which rely on ex vivo handling zygotes require recipient animals for embryo transfer. In contrast, avoids these technically difficult...

Significance The sperm−oocyte fusion step is important to transport the male genome into oocytes. So far, IZUMO1 and FIMP have been identified as fusion-related proteins in spermatozoa, but molecular mechanisms underpinning all of required for this essential process remain unclear. In study, using CRISPR-Cas9−mediated gene knockouts mice, we discover that sperm SOF1, TMEM95, SPACA6 are fertility. As these genes conserved among mammals including human, they may explain not only also...

Renal fibrosis is accompanied by the progression of chronic kidney disease. Despite a number past and ongoing studies, our understanding underlying mechanisms remains elusive. Here we explored renal using mouse model unilateral ureter obstruction. We found that in initial stage damage, where extracellular matrix was not yet deposited, proximal tubular cells arrested at G2 cell cycle. Further analyses indicated cyclin-dependent kinase inhibitor p21 partially involved arrest after damage. A...

Recent progress in development of the CRISPR/Cas9 system has been shown to be an efficient gene-editing technology various organisms. We recently developed a novel method called Genome-editing via Oviductal Nucleic Acids Delivery (GONAD) mice; vivo genome editing that does not require ex handling embryos, and this is newly renamed as "improved GONAD" (i-GONAD). However, limited only mice. Therefore study, we challenge apply rats. Here, determine most suitable condition for gene delivery...

Abstract Background Adenosine deaminase domain containing 2 (ADAD2) is a testis‐specific protein composed of double‐stranded RNA binding and non‐catalytic adenosine domain. A recent study showed that ADAD2 indispensable for the male reproduction in mice. However, detailed functions remain elusive. Objectives This aimed to investigate cause sterility Adad2 mutant mice understand molecular ADAD2. Materials methods homozygous mouse lines, –/– Δ/Δ , were generated by CRISPR/Cas9. Western...

Meiosis is a cell division process with complex chromosome events where various molecules must work in tandem. To find meiosis-related genes, we screened evolutionarily conserved and reproductive tract-enriched genes using the CRISPR/Cas9 system identified potassium channel tetramerization domain containing 19 ( Kctd19 ) as an essential factor for meiosis. In prophase I, deficiency did not affect synapsis or DNA damage response, chiasma structures were also observed metaphase I spermatocytes...

Cilia and flagella are ancient structures that achieve controlled motor functions through the coordinated interaction based on microtubules some attached projections. Radial spokes (RSs) facilitate beating motion of these organelles by mediating signal transduction between dyneins a central pair (CP) singlet microtubules. RS complex isolation from Chlamydomonas axonemes enabled detection 23 radial spoke proteins (RSP1-RSP23), although roles remain unknown. Recently, RSP15 has been reported...

Sperm morphogenesis is a tightly regulated differentiation process, disruption of which leads to sperm malfunction and male infertility. Here, we show that Tex38 knockout (KO) mice are infertile. KO spermatids exhibit excess retention residual cytoplasm around the head, resulting in abnormal morphology with backward head bending. TEX38 interacts colocalizes ZDHHC19, testis-enriched acyltransferase catalyzing protein S-palmitoylation, at plasma membrane spermatids. ZDHHC19 each downregulated...

In the brewing of high-quality sake such as Daiginjo-shu, cerulenin-resistant yeast strains with high producing ability to flavor component ethyl caproate have been used widely. Genetic stability would be important for maintenance both fermentation properties and quality sake. eukaryotes, checkpoint mechanisms ensure genetic stability. However, integrity these in has not examined yet. Here, we investigated yeasts, results suggested that a currently had defect spindle assembly (SAC). We also...

Recent advances in the CRISPR/Cas9 system have demonstrated it to be an efficient gene-editing technology for various organisms. Laboratory mice and rats are widely used as common models of human diseases; however, current standard method create genome-engineered animals is laborious involves three major steps: isolation zygotes from females, ex vivo micromanipulation zygotes, implantation into pseudopregnant females. To circumvent this, we recently developed a novel named Genome-editing via...

In eukaryotes, cell polarity is essential for proliferation, differentiation, and development. It regulated in 3 steps: establishment, maintenance, transition. Compared to current knowledge of establishment the mechanism regulating transition poorly understood. fission yeast during G2 phase, growth undergoes a dramatic transition, from monopolar bipolar (termed NETO: new end take off). this study, we screened systematically protein kinases related NETO using genome-wide kinase deletion...

Abstract Globozoospermia (sperm with an abnormally round head shape) and asthenozoospermia (defective sperm motility) are known causes of male infertility in human patients. Despite many studies, the molecular details globozoospermia etiology still poorly understood. Serine-rich single-pass membrane protein 1 (Ssmem1) is a conserved testis-specific gene mammals. In this study, we generated Ssmem1 knockout (KO) mice using CRISPR/Cas9 system, demonstrated that essential for fertility mice,...

Abstract Alport syndrome is an inherited chronic human kidney disease, characterized by glomerular basement membrane abnormalities. This disease caused mutations in COL4A3 , COL4A4 or COL4A5 gene. The knockout mice for Col4 α 3 4 and 5 are developed well the study of syndrome. However, progression effects pharmacological therapy depend on genetic variability. model was reliable only to mouse. In this study, we created a novel rat utilizing rGONAD technology, which generated with deletion...

In eukaryotes, cell morphogenesis is regulated coordinately with the cycle. fission yeast, network MOR (morphogenesis Orb6 network) consists of 5 conserved proteins, Pmo25, Nak1, Mor2, Orb6, and Mob2, essential for polarity control separation following cytokinesis. Here we show that leucine-rich repeat protein Lrp1 required as a newly recognized component MOR. has 4 repeats in its N-terminus homolog budding yeast Sog2, which RAM (regulation Ace2 activity cellular morphogenesis). was both...

How cell morphology and the cycle are coordinately regulated is a fundamental subject in biology. In fission yeast, 2 germinal center kinases (GCKs), Sid1 Nak1, play an essential role septation/cytokinesis separation/cell polarity control, respectively, as components of septation initiation network (SIN) morphogenesis Orb6 (MOR). Here we show that third GCK, Ppk11, also required for efficient separation particularly, at high temperature. Although Ppk11 not division, this kinase plays...

Cell polarity is essential for various cellular functions during both proliferative and developmental stages, it displays dynamic alterations in response to intracellular extracellular cues. However, the molecular mechanisms underlying spatiotemporal control of transition are poorly understood. Here, we show that fission yeast Cki3 (a casein kinase 1γ homolog) a critical regulator ensure persistent monopolar growth S phase. Unlike wild type, cki3 mutant cells undergo bipolar when phase...

Monoclonal antibodies have been applied in a wide range of biological and medical studies since the advent cell fusion technology. Although techniques improved by using myelomas reagents, researchers still find it difficult to produce monoclonal because long protocols, high costs, low efficiency obtaining hybridomas. To solve these problems, we first developed an iliac lymph node method 1995 rats. In this method, antigen emulsion is injected intramuscularly into tail base, then B lymphocytes...

Microtubule organization and reorganization during the cell cycle are achieved by regulation of number, distribution activity microtubule-organizing centres (MTOCs). In fission yeast, Mto1/2 complex determines cytoplasmic MTOCs. Upon mitosis, microtubule nucleation ceases; inactivation is triggered Mto2 hyperphosphorylation. However, protein kinase(s) that phosphorylates remains elusive. Here we show a conserved signalling network, called MOR (morphogenesis Orb6 network) in negatively...

The correct localization of proteins is linked to their cellular function.

Abstract Tubulointerstitial fibrosis is a progressive process affecting the kidneys, causing renal failure that can be life-threatening. Thus, has become serious concern in ageing population; however, fibrotic development cannot diagnosed early and assessed noninvasively both patients experimental animal models. Here, we found serum amyloid A3 (Saa3) expression potent indicator of fibrosis; also established vivo Saa3/C/EBPβ-promoter bioluminescence imaging as sensitive specific tool for...

Autosomal dominant polycystic kidney disease (ADPKD) is a renal disorder caused by mutations in the PKD2 gene, which encodes polycystin-2/Pkd2, transient receptor potential channel. The precise role of Pkd2 cyst formation remains unclear. fission yeast Schizosaccharomyces pombe has putative channel, Pkd2, shares similarities with human Pkd2. In this study, truncation analyses were conducted to investigate its localization and function. results revealed that localizes not only plasma membrane...

Fission yeast undergoes growth polarity transition from monopolar to bipolar during G 2 phase, designated NETO (New End Take Off). It is known that onset involves two prerequisites, the completion of DNA replication and attainment a certain cell size. However, molecular mechanism remains unexplored. Here, we show casein kinase 1γ, C ki3 critical determinant onset. Not only did cki3 ∆ cells undergo 1‐ or S ‐phase, but they also displayed premature under unperturbed conditions with smaller...

We report a case of unusual annular erythema associated with myelodysplastic syndrome (MDS). A 58-year-old male MDS developed on his back, scaly the dorsa hands, and exsudative eyelids. Histological examination revealed mononuclear cell infiltrate around vessels follicles in mid- to lower dermis. He had no history treatment granulocyte-colony-stimulating factor (G-CSF). Serum granulocyte-macrophage-colony-stimulating (GM-CSF) level was slightly elevated (5.84 pg/ml, normal <2.0 pg/ml),...

Abstract Calcineurin, which is a Ca2+/calmodulin-dependent protein phosphatase, key mediator in calcium signaling diverse biological processes and of clinical importance as the target immunosuppressant FK506. To identify mutant(s) calcineurin activated, inhibiting cellular growth result, we screened for whose temperature sensitivity would be suppressed by FK506 from budding yeast non-essential gene deletion library. We found that cells conserved Verprolin VRP1 had been deleted, required...