Biology subjects

Okamura, K.

Publications and source records attributed to Okamura, K..

10 recordsLinked to original sources

bioRxiv2025

The Knock-In Atlas: A web resource for targeted protein trap by CRISPR/Cas9 in human and mouse cell lines

Various cell engineering techniques have been developed by leveraging the CRISPR-Cas9 technology, but large-scale resources for targeted gene knock-in are still limited. Here we introduce the Knock-in Atlas, a web resource for gene tagging by fluorescent proteins by inserting artificial exons in target gene introns. To produce knock-in cells efficiently and reproducibly, we carefully chose and catalogued guide RNAs (gRNAs) for targeting genes in the human and mouse genomes by taking the gRNA efficacy scores and protein structures around the insertion sites into account. As of August 2025, we have characterized knock-in cell lines for 350 proteins, with a focus on RNA binding proteins, by flow cytometry and confocal microscopy. The transfection and flow cytometry protocols were optimized for several cell lines including HEK293T, eHAP1, HeLa, THP-1, Neuro2a, mouse embryonic fibroblast (MEF) and mouse embryonic stem cell (mESC). A website has been launched to organize the results of initial characterization including flow cytometry data after transfection, confocal microscopy, and western blot results for the genes for which knock-in HEK293T cell lines were already made. The site also provides a database to organize the information of pre-designed gRNAs for the human and mouse genomes. .

molecular biology↗

bioRxiv2024

High Shear Stress Reduces ERG Causing Endothelial-Mesenchymal Transition and Pulmonary Arterial Hypertension

Pathological high shear stress (HSS, 100 dyn/cm2) is generated in distal pulmonary arteries (PA) (100-500 m) in congenital heart defects and in progressive PA hypertension (PAH) with inward remodeling and luminal narrowing. Human PA endothelial cells (PAEC) were subjected to HSS versus physiologic laminar shear stress (LSS, 15 dyn/cm2). Endothelial-mesenchymal transition (EndMT), a feature of PAH not previously attributed to HSS, was observed. H3K27ac peaks containing motifs for an ETS-family transcription factor (ERG) were reduced, as was ERG-Kruppel-like factors (KLF)2/4 interaction and ERG expression. Reducing ERG by siRNA in PAEC during LSS caused EndMT; transfection of ERG in PAEC under HSS prevented EndMT. An aorto-caval shunt was preformed in mice to induce HSS and progressive PAH. Elevated PA pressure, EndMT and vascular remodeling were reduced by an adeno-associated vector that selectively replenished ERG in PAEC. Agents maintaining ERG in PAEC should overcome the adverse effect of HSS on progressive PAH.

cell biology↗

bioRxiv2023

Differentiation of Large Extracellular Vesicles in Oral Fluid: Combined Protocol of Small Force Centrifugation and Pattern Analysis

Extracellular vesicles (EVs) in biofluids are highly heterogeneous entities in terms of their origins and physicochemical properties. Considering the application of EVs in diagnostic and therapeutic fields, it is of extreme importance to establish differentiating methods by which focused EV subclasses are operationally defined. Several differentiation protocols have been proposed; however, they have mainly focused on smaller types of EVs, and the heterogeneous nature of large EVs has not yet been fully explored. In this report, to classify large EVs into subgroups based on their physicochemical properties, we have developed a protocol, named EV differentiation by sedimentation patterns (ESP), in which entities in the crude large EV fraction are first moved through a density gradient of iodixanol with small centrifugation forces, and then the migration patterns of molecules through the gradients are analyzed using a non-hierarchical data clustering algorithm. Based on this method, proteins in the large EV fractions of oral fluids clustered into three groups: proteins shared with small EV cargos and enriched in immuno-related proteins (Group 1), proteins involved in energy metabolism and protein synthesis (Group 2), and proteins required for vesicle trafficking (Group 3). These observations indicate that the physiochemical properties of EVs, which are defined through low-speed gradient centrifugation, are well associated with their functions within cells. This protocol enables the detailed subclassification of EV populations that are difficult to differentiate using conventional separation methods.

molecular biology↗

bioRxiv2021

RNA-dependent RNA polymerases in the black-legged tick produce Argonaute-dependent small RNAs and regulate genes

Small regulatory RNAs (sRNAs) are involved in antiviral defense and gene regulation. Although roles of RNA-dependent RNA Polymerases (RdRPs) in sRNA biology are extensively studied in nematodes, plants and fungi, understanding of RdRP homologs in other animals is still lacking. Here, we study sRNAs in the ISE6 cell line, which is derived from the black-legged tick, an important vector of human and animal pathogens. We find abundant classes of [~]22nt sRNAs that require specific combinations of RdRPs and sRNA effector proteins (Argonautes or AGOs). RdRP-dependent sRNAs possess 5- monophosphates and are mainly derived from RNA polymerase III-transcribed genes and repetitive elements. Knockdown of RdRPs misregulates genes including RNAi-related genes and the regulator of immune response Dsor1. Sensor assays demonstrate that Dsor1 is downregulated by RdRP through the 3UTR that contains a target site of RdRP-dependent repeat-derived sRNAs. Consistent with viral gene repression by the RNAi mechanism using virus-derived small interfering RNAs, viral transcripts are upregulated by AGO knockdown. On the other hand, RdRP knockdown unexpectedly results in downregulation of viral transcripts. This effect is dependent on Dsor1, suggesting that antiviral immunity is enhanced by RdRP knockdown through Dsor1 upregulation. We propose that tick sRNA pathways control multiple aspects of immune response via RNAi and regulation of signaling pathways. Author summaryRNA-dependent RNA Polymerases (RdRPs) are essential for biogenesis of small regulatory RNAs (sRNAs) in many organisms such as plants and fungi, but its general importance in animals besides nematodes remains controversial because experimental evidence is lacking. By using a tick cell line, we demonstrate that RdRP-dependent sRNAs are abundantly expressed and tick RdRPs regulate gene expression. These results indicate that ticks have unexpectedly complex sRNA biogenesis pathways that are essential for proper gene regulation. Interestingly, we show that RdRP negatively regulates immune response by changing expression of a gene that is essential in an immunity-related signaling pathway. Because ticks are important vectors of human and animal pathogens, studying the novel tick sRNA pathways and their effects on immune signaling may lead to a better understanding of vector-virus interactions.

molecular biology↗

bioRxiv2021

Pseudoautosomal gene SHOX exhibits sex-biased random monoallelic expression and contributes to sex difference in height

Adult men are, on average, [~]13 cm taller than adult women. Although previous studies have suggested a significant contribution of sex chromosomal genes to sexual dimorphism in height, all attempts to identify a male-specific growth gene have failed. In the present study, we analyzed transcripts from cartilage tissues, and found that the expression of SHOX, a growth-promoting gene in the pseudoautosomal region on the X and Y chromosomes, was lower in females than in males. DNA methylation analyses showed that SHOX has some characteristics of genes subjected to X chromosome inactivation (XCI). These findings indicate that sex difference in human height is mainly ascribed to incomplete spreading of XCI on a pseudoautosomal gene. More importantly, RT-PCR of fibroblast clones revealed XCI-independent random clonal monoallelic expression of SHOX. We presume that during eutherian evolution, SHOX translocated from an autosome to the proto-sex chromosome without losing the epigenetic memory of random clonal monoallelic expression and subsequently underwent partial XCI. This study provides a novel model of epigenetic gene regulation leading to phenotypic diversity in humans.

genetics↗

bioRxiv2020

Collection of 2429 constrained headshots of 277 volunteers for deep learning

Deep learning has rapidly been filtrating many aspects of human lives. In particular, image recognition by convolutional neural networks has inspired numerous studies in this area. Hardware and software technologies as well as large quantities of data have contributed to the drastic development of the field. However, the application of deep learning is often hindered by the need for big data and the laborious manual annotation thereof. To experience deep learning using the data compiled by us, we collected 2429 constrained headshot images of 277 volunteers. The collection of face photographs is challenging in terms of protecting personal information; we established an online procedure in which both the informed consent and image data could be obtained. We did not collect personal information, but issued agreement numbers to deal with withdrawal requests. Gender and smile labels were manually and subjectively annotated only from the appearances, and final labels were determined by majority among our team members. Rotated, trimmed, resolution-reduced, decolorized, and matrix-formed data were allowed to be publicly released. Moreover, simplified feature vectors for data sciences were released. We performed gender recognition by building convolutional neural networks based on the Inception V3 model with pre-trained ImageNet data to demonstrate the usefulness of our dataset.

animal behavior and cognition↗

bioRxiv2020

RT-qPCR analyses on the osteogenic differentiation from human iPS cells: An investigation of reference genes

Reverse transcription quantitative PCR (RT-qPCR) is used to quantify gene expression and require standardization with reference genes. We sought to identify the reference genes best suited for experiments that induce osteogenic differentiation from human induced pluripotent stem (iPS) cells. They were cultured in an undifferentiated maintenance medium and after confluence, further cultured in an osteogenic differentiation medium for 28 days. RT-qPCR was performed on undifferentiation markers, osteoblast and osteocyte differentiation markers, and reference gene candidates. The expression stability of each reference gene candidate was ranked using four algorithms. General rankings identified TATA box binding protein (TBP) in the first place, followed by transferrin receptor (TFRC), ribosomal protein large P0 (RPLP0), and finally, beta-2-microglobulin (B2M), which was revealed as the least stable. Interestingly, universally used GAPDH and ACTB were found to be unsuitable. Our findings strongly suggest a need to evaluate the expression stability of reference gene candidates for each experiment.

cell biology↗

bioRxiv2020

Phosphorylation of the Anaphase Promoting Complex activator CDH1/FZR regulates the transition from Meiosis I to Meiosis II in mouse male germ cell

CDH1/FZR is an activator of Anaphase promoting complex/Cyclosome (APC/C), best known for its role as E3 ubiquitin ligase that drives the cell cycle. APC/C activity is regulated by CDK-mediated phosphorylation of CDH1 during mitotic cell cycle. Although the critical role of CDH1 phosphorylation has been shown mainly in yeast and in vitro cell culture studies, its biological significance in mammalian tissues in vivo remained elusive. Here, we examined the in vivo role of CDH1 phosphorylation using a mouse model, in which non-phosphorylatable substitutions were introduced in the putative CDK-phosphorylation sites of CDH1. Although ablation of CDH1 phosphorylation did not show substantial consequences in mouse somatic tissues, it led to severe testicular defects resulting in male infertility. In the absence of CDH1 phosphorylation, male juvenile germ cells entered meiosis normally but skipped meiosis II producing diploid spermatid-like cells. In aged testis, male germ cells were overall abolished, showing Sertoli cell-only phenotype. The present study demonstrated that phosphorylation of CDH1 is required for temporal regulation of APC/C activity at the transition from meiosis I to meiosis II, and for spermatoginial stem cell maintenance, which raised an insight into the sexual dimorphism of CDH1-regulation in germ cells.

cell biology↗

bioRxiv2020

A meiosis-specific factor MRM/C19orf57 modulates localization of RAD51 and DMC1 recombinases to DSBs in mouse meiotic recombination

Meiotic recombination is critical for genetic exchange and generation of chiasmata that ensures faithful chromosome segregation during meiosis I. Meiotic recombination is initiated by DNA double-strand break (DSB) followed by multiple processes of DNA repair. The exact mechanisms how recombinases localize to DSB remained elusive. Here we show that C19orf57/4930432K21Rik/BRME1 is a new player for meiotic recombination in mice. C19orf57/4930432K21Rik/BRME1 associates with ssDNA binding proteins, BRCA2 and MEILB2/HSF2BP, critical recruiters of recombinases onto DSB sites. Disruption of C19orf57/4930432K21Rik/BRME1 shows severe impact on DSB repair and male fertility. Remarkably, removal of single stranded DNA (ssDNA) binding proteins from DSB sites is delayed, and reciprocally the loading of RAD51 and DMC1 onto resected ssDNA is impaired in Brme1 KO spermatocytes. We propose that C19orf57/4930432K21Rik/BRME1 modulates localization of recombinases to meiotic DSB sites through the interaction with the BRCA2-MEILB2/HSF2BP complex during meiotic recombination.

molecular biology↗

bioRxiv2016

Fetal therapy model of myelomeningocele with three-dimensional skin using amniotic fluid cell-derived induced pluripotent stem cells

Myelomeningocele (MMC) is a congenital disease without genetic abnormalities. Neurological symptoms are irreversibly impaired after birth. No effective treatment has been reported to date. Only surgical repairs have reported so far. In this study, we performed antenatal treatment of MMC with an artificial skin using induced pluripotent stem cells (iPSCs) generated from a patient with Down syndrome (AF-T21-iPSCs) and twin-twin transfusion syndrome (AF-TTTS-iPSCs) to a rat model. We manufactured three-dimensional skin with epidermis generated from keratinocytes derived from AF-T21-iPSCs and AF-TTTS-iPSCs and dermis of human fibroblasts and collagen type I. For generation of epidermis, we developed a novel protocol using Y-27632 and epidermal growth factor. The artificial skin was successfully covered over MMC defect sites during pregnancy, implying a possible antenatal surgical treatment with iPSC technology.

cell biology↗