Biology subjects

Suzuki, H.

Publications and source records attributed to Suzuki, H..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

mm2-ivh: simple and precise overlap detection in alpha satellite HORs with interval hashing

SummaryWe propose a new algorithm, "interval hashing," which distinguishes identical k-mers arising from different repeat sequences, particularly in complex repeat arrays such as alpha satellite HORs. We implement this algorithm as a fork of minimap2, named mm2-ivh. In local assembly of alpha satellite HORs, mm2-ivh accurately reconstructs more haplotypes than assemblers using standard minimizers. Availabilitymm2-ivh is available under the MIT license at https://github.com/ocxtal/mm2-ivh and runs on common Unix-compatible systems.

bioinformatics↗

bioRxiv2025

A simple and versatile plasma membrane staining method for visualizing living cell morphology in reproductive tissues across diverse plant species

Plant reproduction involves dynamic spatiotemporal changes that occur deep within maternal tissues. In ovules of Arabidopsis thaliana (A. thaliana), one of the two synergid cells degenerates at fertilization, while the fertilized egg cell (zygote) undergoes directional elongation followed by asymmetric division to initiate embryonic patterning. However, morphological analysis of these events has been hampered by the limitations of conventional cell wall staining, which fails to label cells lacking complete walls, and by the requirement for transgenic fluorescent reporters to visualize cell outlines. Here, we report that the membrane-specific fluorescent dye FM4-64 readily permeates ovules, allowing clear visualization of reproductive cell morphology both before and after fertilization. This staining method supports high-resolution time-lapse imaging and quantitative analysis of early embryogenesis in living tissues. Importantly, it is applicable not only to the angiosperm A. thaliana but also to the liverwort Marchantia polymorpha (M. polymorpha) and the fern Ceratopteris richardii (C. richardii), enabling the visualization of live reproductive cell structures within maternal tissues and revealing fertilization-associated morphological changes. This simple and robust method thus provides a valuable tool for spatiotemporal and quantitative analyses of reproductive processes across a broad range of plant species, without the need to generate transgenic lines.

plant biology↗

bioRxiv2025

Hepatocyte-specific CLSTN3B ablation impairs lipid droplet maturation and alleviates diet-induced steatohepatitis in mice

Excessive lipid accumulation in hepatocytes, a hallmark of metabolic dysfunction-associated steatotic liver disease (MASLD), can lead to progressive liver damage. Understanding the molecular mechanisms governing lipid storage in hepatocytes is essential for identifying therapeutic targets to halt MASLD progression. Here, we show a pivotal role for the protein calsyntenin 3{beta} (CLSTN3B) in promoting lipid droplet (LD) maturation and lipid storage in hepatocytes. Previously characterized as an endoplasmic reticulum (ER)-LD contact protein that facilitates LD maturation in adipocytes, we now show that CLSTN3B expression is strongly induced in mouse hepatocytes by peroxisome proliferator-activated receptor gamma (PPAR{gamma}) in response to dietary caloric excess. Hepatocyte-specific deletion of CLSTN3B in mice significantly increases energy expenditure, reduces metabolic efficiency, and protects against diet-induced hepatic steatosis and fibrosis. Mechanistically, CLSTN3B deficiency causes reduced LD phospholipid coverage and increased lipase recruitment. This results in enhanced fatty acid oxidation driven by a futile cycle of lipolysis and re-esterification. Notably, human clinical data reveal a positive correlation between hepatic CLSTN3B expression and MASLD severity and progression, emphasizing its relevance to human disease. Together, our findings establish CLSTN3B as a key regulator of hepatocyte lipid storage and metabolic efficiency and highlight its potential as a therapeutic target in MASLD.

physiology↗

bioRxiv2025

Acquisition and repeated alteration of (TTGGG)n telomeric repeats in Odonata (dragonflies and damselflies)

Chromosome ends of most eukaryotes are composed of simple telomeric repeats. For arthropods, TTAGG pentanucleotide repeats, (TTAGG)n has been considered as the ancestral telomeric repeat. However, in the order Odonata, the earliest diverged group in insects that contains dragonflies and damselflies, (TTAGG)n signal has been almost undetectable in most examined species. Here, we report a unique pentanucleotide repeat (TTGGG)n as the typical telomeric repeat sequence for Odonata (dragonflies and damselflies). Based on genomic information from ten Odonata species, (TTGGG)n was considered the most likely candidate for telomeric repeat sequences. By fluorescence in situ hybridization (FISH) using 12 Odonata species, clear (TTGGG)n signals were detected at the chromosome ends in both dragonflies and damselflies. By Southern hybridization using 63 Odonata species, strong (TTGGG)n signals were detected from the majority of species covering all three suborders of Odonata, indicating that the telomeric repeat of the common ancestor of extant Odonata is (TTGGG)n. Notably, there were a few Odonata species in which (TTGGG)n signals were faint or absent, suggesting that the telomeric repeat sequence has repeatedly diverged in Odonata, even within genera such as Sympetrum. We also identified telomerase genes in both dragonflies and damselflies, and in some species, more than two telomerase genes are suggested to be present. Overall, this study demonstrates the ancestral acquisition of novel telomeric repeats and their repeated alteration in Odonata.

cell biology↗

bioRxiv2025

Human TIMELESS, a potential circadian clock regulator, plays an essential role in survival and reawakening of metastasis-initiating cells in bone

Bone metastasis is becoming increasingly common globally. In such cases, cancer cells disseminate from the primary site to bone, subsequently entering a dormant state. Following a certain incubation period, the cells reawaken and grow, forming a metastatic mass. These dormant cells are called metastasis-initiating cells (MICs), but their survival and reawakening are poorly understood. Here, we established an in vivo MIC-reawakening mouse model in bone. Microarray analysis demonstrated enrichment for mitochondrial oxidative phosphorylation (OXPHOS) and fatty acid synthesis in isolated MICs. In addition, transcriptional regulator TIMELESS was identified as an independent poor prognostic factor by using clinical transcriptomic datasets, which was validated by immunostaining on 209 breast cancer cases. TIMELESS-deficient breast, prostate, and bladder cancer cell lines exhibited reduced viability and MIC reawakening in bone. Single-cell analysis on the epigenetic landscape of MICs revealed that motif activities of CLOCK and BMAL1/ARNTL were enhanced in a cluster with increased TIMELESS accessibility scores. As TIMELESS regulates stemness through the OXPHOS metabolic state, an inhibitor targeting the metabolic pathway, MP-A08, was identified to suppress MIC reawakening and growth in bone more efficiently than cisplatin. These results suggest that TIMELESS regulates survival and reawakening in MICs and MP-A08 could contribute to adjuvant therapeutic strategies.

cancer biology↗

bioRxiv2025

Synergistic RAS-MAPK and AKT Activation in MYC-Driven Tumors via Adjacent PVT1 Rearrangements

MYC-driven (MYC+) cancers are aggressive and often fatal. MYC dysregulation is a key event in these cancers, but overexpression of MYC alone is not always enough to cause cancer. Plasmocytoma Variant Translocation 1 (PVT1), a long non-coding RNA (lncRNA) adjacent to MYC on chromosome 8 is a rearrangement hotspot in many MYC+ cancers. In addition to being co-amplified with MYC, the genomic rearrangement at PVT1 involves translocation, which has had obscure functional consequences. We report that translocation at the PVT1 locus cause asymmetric enrichment of 5-PVT1 and loss of 3-PVT1. Despite being classified as a non-coding RNA, the retained 5 region of PVT1 generates a circular RNA (CircPVT1) that codes for the novel peptide we call Firefox (FFX). FFX augments AKT signaling and synergistically activates MYC and mTORC1 in these cells. Further, the 3 end of PVT1, which is lost during the translocation, codes for a tumor-suppressing micropeptide we named as Honeybadger (HNB). We demonstrate that HNB interacts with KRAS and disrupts the activation of KRAS effectors. Loss of HNB leads to activation of RAS/MAPK signaling pathway, and enhances MYC stability by promoting phosphorylation of MYC at Ser62. These findings identify PVT1 as a critical node that synchronizes MYC, AKT, and RAS-MAPK activities in cancer. Our study thus identifies a key mechanism by which rearrangements at the PVT1 locus activate additional oncogenic pathways that synergize with MYC to exacerbate the aggressiveness of MYC+ cancers. This newfound understanding explains the poor prognosis associated with MYC+ cancers and offers potential therapeutic targets that could be leveraged in treatment strategies for these cancers.

cancer biology↗

bioRxiv2024

Implication of S-1 biomarkers on aging and prognosis in elderly patients with lung cancer treated by adjuvant S-1 chemotherapy: Accompanying results of the Setouchi Lung Cancer Group Study 1201

BACKGROUNDSThe management of elderly patients presents several challenges due to age-related declines; however recent recommendations advocate for age not being the sole determinant for adjuvant treatment decisions in patients with non-small cell lung cancer (NSCLC). Aging may alter expression levels of 5-fluorouracil (5-FU) biomarkers. METHODSExpression changes with aging were explored using The Cancer Genome Atlas (TCGA) database. 5-FU-related biomarker expressions, including thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyltransferase, epidermal growth factor receptor (EGFR), and excision repair cross-complementation group-1 (ERCC1), were assessed by the quantitative reverse-transcription PCR assays in 89 NSCLCs elderly patients ([≥] 75 years old) receiving adjuvant S-1, an oral fluoropyrimidine agent, therapy in a SLCG1201 trial. RESULTS: TCGA database analysis (n=955) indicated decreased TS expression with aging, particularly in those 75 or older. In the SCLG1201 trial, univariate analysis revealed that high EGFR and low TS expressions correlated with favorable recurrence-free survival (RFS)(p=0.0264) and overall survival (OS)(p=0.0365), respectively. Multivariable analysis confirmed pathological stage as an independent prognostic factor for both RFS and OS (p=0.0052 and 0.0352, respectively). EGFR mutant tumors (n=27) showed significantly higher DPD (p=0.0066) and EGFR (p<0.0001) expressions, and lower TS(p=0.0125) and ERCC1(p=0.0015) expressions. CONCLUSIONDespite pathological stage being an independent prognostic factor, high EGFR and low TS expressions may predict better clinical outcomes in elderly NSCLC patients using adjuvant S-1. The age-related decrease in TS expression supports the potential benefit of 5-FU-based therapy in them compared to younger patients. Further research is warranted to validate these clinical implications.

pharmacology and toxicology↗

bioRxiv2024

Analysis of the high-order conformational changes in glyceraldehyde-3-phosphate dehydrogenase induced by nicotinamide adenine dinucleotide, adenosine triphosphate, and oxidants

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the sixth step of glycolysis. As a moonlighting protein, GAPDH interacts with other molecules, seemingly unrelated to glycolysis, to exert additional functions, such as inducing apoptosis. However, specific mechanisms underlying these interactions remain unclear. Therefore, in this study, we aimed to elucidate these mechanisms by analyzing human and porcine GAPDHs based on their three-dimensional (3D) structures using various biophysical techniques, including nuclear magnetic resonance spectroscopy, gel filtration chromatography, and thermal shift assay. Although GAPDH became unstable when nicotinamide adenine dinucleotide (NAD+) was removed (apo state), the 3D structure of the tetramer was maintained regardless of the temperature. However, in the presence of adenosine triphosphate (ATP), GAPDH split into dimers at low temperatures, exposing the interface between the dimers and increasing the flexibility of the side chains at the site. Moreover, subunit splitting also occurred upon exposure to oxidizing agents, such as hydrogen peroxide. These findings suggest that GAPDH maintains a stable tetramer in the presence of NAD+ but becomes unstable and easily oxidized upon NAD+ depletion. When multiple residues, including those other than the cysteine residue at the active site, are oxidized by reactive oxygen species or nitric oxide, or when it interacts with ATP, GAPDH splits into dimers. This subunit splitting may trigger interactions with other molecules.

biochemistry↗

bioRxiv2024

AistSeq: An In-House Tn5-Based Plasmid Sequencing Platform Using A Compact Benchtop Sequencer

Sequence verification of plasmids is a fundamental process in synthetic biology. For plasmid sequence verification using next-generation sequencing (NGS) library preparation, Tn5 transposase is widely used. Streamlined sequencing workflow for laboratory-scale applications is important; however, recombinant Tn5 production in-house can be laborious. In this study, we demonstrated that the addition of a large soluble tag was not essential for purification and that the fusion of a His10 tag and protein A was sufficient to purify sufficient amounts of active Tn5 transposase. In addition, we evaluated exonuclease-based genomic DNA digestion for plasmid sequencing from an E. coli lysate and the data analysis pipeline of sequences derived from the Illumina iSeq100 platform for de novo assembly, reference mapping, and annotation. This study proposes a simple workflow of an in-house Tn5-based plasmid Sequencing platform using a compact benchtop sequencer (AistSeq). Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=37 SRC="FIGDIR/small/618112v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@f1546corg.highwire.dtl.DTLVardef@1dd947aorg.highwire.dtl.DTLVardef@3efb78org.highwire.dtl.DTLVardef@efbde1_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

INCOMPATIBILITY GROUPS OF PSEUDOMONAS PLASMIDS REVISITED: COMPREHENSIVE ANALYSIS OF R-FACTORS AND THEIR REPLICONS

Plasmids are the most influential engines of bacterial evolution and horizontal gene transfer, fueling the global spread of traits such as antimicrobial resistance. Their deep evolutionary relationships, however, remain difficult to resolve because current classification schemes are constrained by host range and nucleotide similarity. Replication initiation proteins (RIPs), which govern plasmid persistence and diversification, also remain poorly annotated across public databases. Here we establish PInc, a curated and experimentally grounded replicon classification framework anchored in historically defined incompatibility groups of Pseudomonas plasmids. Homology searches beyond PInc revealed that most replication initiators analyzed here share a conserved winged-helix (WH) domain, defining a broad WH RIP superfamily. Using the conserved WH region, we reconstructed a large-scale phylogeny that linked WH RIPs to over 100,000 plasmids, representing approximately half of those analyzed across public databases. This phylogeny resolved eight major clades and the deep split between the single- and double-winged-helix superclades, while revealing clade-specific host and environmental distributions and substantial RIP diversity not captured by current typing tools or annotation schemes. Together, these results overcome the historical host bias of plasmid typing and provide a replication-centered view of plasmid diversification across bacterial lineages and environments.

microbiology↗

bioRxiv2024

KAI2-dependent signaling controls vegetative reproduction in Marchantia polymorpha through activation of LOG-mediated cytokinin synthesis

Marchantia polymorpha reproduces vegetatively (asexually) by producing propagules known as gemmae within gemma cups and sexually through spores. We previously reported that KARRIKIN INSENSITIVE 2 (KAI2)-dependent signaling promotes gemma cup and gemma formation. KAI2A perceives unidentified endogenous ligand(s), tentatively referred to as KAI2 ligands (KL). Perception of KL by KAI2 triggers MAX2-dependent proteolysis of MpSMXL. In this study, we identified genes working downstream of KAI2-dependent signaling in M. polymorpha. We found that KAI2-dependent signaling positively controls the expression of MpLONLEY GUY (MpLOG), encoding a cytokinin biosynthesis enzyme. Disruption of the MpLOG function decreased endogenous cytokinin levels and caused defects similar to KAI2-dependent signaling mutants. Moreover, supplying exogenous cytokinins rescued the defects of Mplog and KAI2-dependent signaling mutants, implying that cytokinins work downstream of KAI2-dependent signaling. Activation of MpLOG by KAI2-dependent signaling occurs in a highly cell-type-specific manner, leading to cell-specific induction of GEMMA CUP-ASSOCIATED MYB1 (GCAM1), the master regulator of vegetative reproduction of M. polymorpha. We propose a genetic cascade, starting from KAI2-dependent signaling, that promotes vegetative reproduction through the induction of MpLOG and GCAM1. The interaction between KAI2-dependent signaling and cytokinin in M. polymorpha provides a novel insight into the function and evolution of KAI2-dependent signaling.

plant biology↗

bioRxiv2024

Speech Synthesis from Electrocorticogram During Imagined Speech Using a Transformer-Based Decoder and Pretrained Vocoder

Synthesizing speech from Electrocorticogram (ECoG) signals recorded during imagined speech remains a challenge due to the absence of synchronized audio signals for training. To address this, we propose a training framework that utilizes audio recorded during overt speech tasks as a surrogate ground truth for imagined speech signals, based on the consistency of the linguistic content. We employed a Transformer-based decoder to generate log-mel spectrograms from imagined speech ECoG, which were then converted into waveform audio using a pre-trained Parallel WaveGAN. In experiments involving ECoG recordings from 13 participants, the synthesized speech achieved dynamic time warping-aligned Pearson correlation coefficients ranging from 0.74 to 0.84 with the proxy targets. These results demonstrate that overt speech audio can serve as an effective training target for reconstructing imagined speech, offering a viable solution for training decoders in the absence of behavioral output.

neuroscience↗

bioRxiv2024

Image retrieval based on closed-loop visual-semantic neural decoding

Neural decoding via the latent space of deep neural network models can infer perceived and imagined images from neural activities, even when the image is novel for the subject and decoder. Brain-computer interfaces (BCIs) using the latent space enable a subject to retrieve intended image from a large dataset on the basis of their neural activities but have not yet been realized. Here, we used neural decoding in a closed-loop condition to retrieve images of the instructed categories from 2.3 million images on the basis of the latent vector inferred from electrocorticographic signals of visual cortices. Using a latent space of contrastive language-image pretraining (CLIP) model, two subjects retrieved images with significant accuracy exceeding 80% for two instructions. In contrast, the image retrieval failed using the latent space of another model, AlexNet. In another task to imagine an image while viewing a different image, the imagery made the inferred latent vector significantly closer to the vector of the imagined category in the CLIP latent space but significantly further away in the AlexNet latent space, although the same electrocorticographic signals from nine subjects were decoded. Humans can retrieve the intended information via a closed-loop BCI with an appropriate latent space.

neuroscience↗

bioRxiv2024

Latent mitotic vulnerability of AML cells induced by therapeutic agents

Acute myeloid leukemia (AML) is a hematopoietic malignancy with a poor prognosis. Understanding the unidentified properties of AML cells is beneficial for the identification of novel therapeutic strategies for AML. In this study, we uncover the vulnerabilities of AML cells in mitosis when exposed to therapeutic agents. Through comparative analysis of large-scale data quantifying drug effects on cancer cell proliferation, the drug targeting the cell cycle and mitosis are predicted to possess high cytotoxicity against AML cell lines. Consistently, live-cell imaging with microwell devices demonstrates that clinical drugs targeting the cell cycle processes, such as idarubicin, pevonedistat and vincristine, potently induce mitotic cell death in AML cells. While these therapeutic agents also induce cell death through S/G2 phase arrest, the cytotoxic effects during mitosis are notably more pronounced. Furthermore, by employing additional inhibition of Chk1 to override the G2/M checkpoint, the AML cells stalled in the S/G2 phase prematurely enter mitosis, resulting in a significant increase in cell death. Collectively, these results unveiled the latent mitotic vulnerabilities of AML cells, providing a basis for developing novel therapeutic interventions.

cancer biology↗

bioRxiv2024

Heteromeric amyloid filaments of ANXA11 and TDP-43 in FTLD-TDP Type C

Neurodegenerative diseases are characterised by the abnormal filamentous assembly of specific proteins in the central nervous system1. Human genetic studies established a causal role for protein assembly in neurodegeneration2. However, the underlying molecular mechanisms remain largely unknown, which is limiting progress in developing clinical tools for these diseases. Recent advances in electron cryo-microscopy (cryo-EM) have enabled the structures of the protein filaments to be determined from patient brains1. All diseases studied to date have been characterised by the self-assembly of a single intracellular protein in homomeric amyloid filaments, including that of TAR DNA-binding protein 43 (TDP-43) in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) Types A and B3,4. Here, we used cryo-EM to determine filament structures from the brains of individuals with FTLD-TDP Type C, one of the most common forms of sporadic FTLD-TDP. Unexpectedly, the structures revealed that a second protein, annexin A11 (ANXA11), co-assembles with TDP-43 in heteromeric amyloid filaments. The ordered filament fold is formed by TDP-43 residues G282/284-N345 and ANXA11 residues L39-L74 from their respective low-complexity domains (LCDs). Regions of TDP-43 and ANXA11 previously implicated in protein-protein interactions form an extensive hydrophobic interface at the centre of the filament fold. Immunoblots of the filaments revealed that the majority of ANXA11 exists as a [~]22 kDa N-terminal fragment (NTF) lacking the annexin core domain. Immunohistochemistry of brain sections confirmed the co-localisation of ANXA11 and TDP-43 in inclusions, redefining the histopathology of FTLD-TDP Type C. This work establishes a central role for ANXA11 in FTLD-TDP Type C. The unprecedented formation of heteromeric amyloid filaments in human brain revises our understanding of amyloid assembly and may be of significance for the pathogenesis of neurodegenerative diseases.

neuroscience↗

bioRxiv2024

Environmental rather than spatial factors determine trait-based community structure: a case study with zooplankton

Ecological community structure, which has traditionally been described in terms of taxonomic units, is driven by a combination of dispersal and environmental filters. Traits have recently been recognized as alternative units for quantifying community parameters, but they may have important differences with taxonomic units. For example, as taxon-based community structures are determined by the identities of individual species, they may be more limited by species dispersal constraints, whereas trait-based community structures may be more regulated by environmental conditions because functional traits should have stronger linkages to habitat types rather than their specific locations. This suggests that the relative importance of dispersal and environmental filters may vary depending on the units employed to define community structure, yet how the contributions of these filters compare remains unclear. Zooplankton in lakes and reservoirs are an ideal system for examining the relative importance of these filters as they have moderate dispersal ability and inhabit communities in isolated habitats. In this study, we examined zooplankton assemblages in 87 artificial reservoirs throughout the Japanese archipelago to test the hypothesis that environmental filters play a more dominant role in determining trait-based community structures than taxon-based structures. To describe trait-based communities, we categorized zooplankton taxa into 12 functional groups using ecologically meaningful traits, including body size, feeding habits and mode, and population growth rate. We then examined the effects of both spatial configuration (reflecting dispersal filters) and environmental variables such as reservoir size and depth, trophic conditions, and fish assemblages for taxon- and trait-based community structures. Although variation in the taxon-based structure was explained equally well by spatial and environmental variables, variation in the trait-based structure explained by environmental variables was nearly twice that of spatial variables. These results support the idea that environmental filters play a more central role in determining trait-based community structures, and show that the relative importance of spatial and environmental filters changes with the way we define community structure. Open research statementAll the data used in this study was obtained from disclosed sources including the Database of Dams in Japan (http://mudam.nilim.go.jp/home) for the location and watershed areas; the Japan Dam Foundation (http://damnet.or.jp/) for the dam specification data; and the River Environmental Database (Red: http://www.nilim.go.jp/lab/fbg/ksnkankyo/index.html) for water chemistry, fish, and zooplankton. Data obtained from local reports of the national reservoir management office, Japan, can be provided by the authors upon request. All analyses were conducted in the R programming language and all functions used in the present study originate from existing packages.

ecology↗

bioRxiv2024

Feasibility of decoding covert speech in ECoG with aTransformer trained on overt speech

Several attempts for speech brain-computer interfacing (BCI) have been made to decode phonemes, sub-words, words, or sentences using invasive measurements, such as the electrocorticogram (ECoG), during auditory speech perception, overt speech, or imagined (covert) speech. Decoding sentences from covert speech is a challenging task. Sixteen epilepsy patients with intracranially implanted electrodes participated in this study, and ECoGs were recorded during overt speech and covert speech of eight Japanese sentences, each consisting of three tokens. In particular, Transformer neural network model was applied to decode text sentences from covert speech, which was trained using ECoGs obtained during overt speech. We first examined the proposed Transformer model using the same task for training and testing, and then evaluated the models performance when trained with overt task for decoding covert speech. The Transformer model trained on covert speech achieved an average token error rate (TER) of 46.6% for decoding covert speech, whereas the model trained on overt speech achieved a TER of 46.3% (p > 0.05; d = 0.07). Therefore, the challenge of collecting training data for covert speech can be addressed using overt speech. The performance of covert speech can improve by employing several overt speeches.

neuroscience↗

bioRxiv2023

Highly efficient transgenic mouse production using piggyBac and its application to rapid phenotyping at the founder generation

Pronuclear microinjection is the most popular method for producing transgenic (Tg) animals. Because the production efficiency is typically less than 20%, phenotypic characterization of Tg animals is generally performed on the next generation (F1) onwards. However, apart from in rodents, in many animal species with long generation times, it is desirable to perform phenotyping in the founder (F0) generation. In this study, we attempted to optimize a method of Tg mouse production to achieve higher Tg production efficiency using piggyBac transposon systems and established optimal conditions under which almost all individuals in the F0 generation were Tg. We also succeeded in generating bacterial artificial chromosome Tg mice with efficiency of approximately 70%. By combining this method with genome editing technology, we established a new strategy to perform phenotyping of mice with tissue-specific knockout using the F0 generation. Taking the obtained findings together, by using this method, experimental research using Tg animals can be carried out more efficiently.

bioengineering↗