Biology subjects

Totsuka, Y.

Publications and source records attributed to Totsuka, Y..

3 recordsLinked to original sources

bioRxiv2026

Dietary phosphorus restriction enables multigenerational breeding of α-Klotho-deficient rats

Severe -Klotho deficiency causes early mortality and reproductive impairment, limiting the maintenance and physiological analysis of rat models. We generated rats carrying a CRISPR/Cas9-mediated -Klotho deletion spanning exons 1-5 and evaluated dietary phosphorus restriction primarily in the Wistar-Imamichi background. Maternal low-phosphorus (LP) feeding from mating through lactation was associated with higher overall survival to weaning (92.6% versus 85.8% under normal-phosphorus conditions) and a higher proportion of KO animals among weaned survivors (34/113 versus 23/145). Maternal LP feeding also reduced plasma phosphorus in KO pups. Continued LP feeding after weaning supported growth, gonadal development, and natural KO x KO breeding. Across 15 litters, 74 of 85 offspring survived to weaning, and KO colonies were maintained to at least the fifth generation. We additionally characterized 24-h metabolic rhythms in LP-maintained KO animals using indirect calorimetry. These profiles remained heterogeneous under a light-dark cycle. In conclusion, dietary phosphorus restriction provides a reliable strategy for the multigenerational maintenance of severe -Klotho-deficient rats, establishing a maintainable model to investigate residual metabolic and physiological phenotypes.

physiology↗

bioRxiv2025

Evaluation of graphene oxide-mediated NET formation using HL-60-derived neutrophil-like cells

Neutrophil extracellular traps (NETs) are chromatin-based structures released by activated neutrophils in response to pathogens or chemical stimuli, contributing to host defense but also implicated in autoimmune disease and inflammation. As NET formation gains attention as an endpoint in in vitro immunotoxicity screening, the lack of reproducible and scalable systems hampers its broader application. Here, we developed an in vitro assay using HL-60-derived neutrophil-like cells (dHL-60) differentiated with all-trans retinoic acid to evaluate NET-inducing activity in a standardized, non-animal model. Graphene oxide (GO), a model nanomaterial known to trigger NETs in primary neutrophils, induced concentration-dependent NET formation in dHL-60 cells, with maximal induction at intermediate doses and attenuation at higher concentrations, likely due to particle aggregation. NET formation was validated by extracellular DNA staining and scanning electron microscopy. GO also induced superoxide-mediated ROS production, as confirmed by electron spin resonance, consistent with canonical NETosis pathways. Furthermore, GO suppressed PMA-induced NET formation, suggesting a dose- and context-dependent immunomodulatory effect. Collectively, our results demonstrate that dHL-60 cells recapitulate key features of NETosis observed in primary neutrophils and provide a practical, reproducible model for investigating immune responses to nanomaterials. This system supports the development of non-animal approaches for assessing immunological effects of chemical substances under controlled in vitro conditions.

pharmacology and toxicology↗

bioRxiv2022

Necessity of integrated genomic analysis to establish a designed knock-in mouse from CRISPR-Cas9-induced mutants

The CRISPR-Cas9 method for generation of knock-in mutations in a rodent embryo yields many F0 generation candidates that may have the designed mutations. Genetic mutations must be confirmed using genomic DNA with a mixture of designed and unexpected mutations. In our study for establishing Prlhr-Venus knock-in reporter mice, we found that genomic rearrangements near the targeted knock-in allele, tandem multicopy at a target allele locus and mosaic genotype of two different knock-in alleles occurred in addition to the designed knock-in mutation in the F0 generation. Conventional PCR and genomic sequencing were not able to detect mosaicism or discriminate between the designed one-copy knock-in mutant and a multicopy-inserted mutant. However, by using a combination of Southern blotting and the next-generation sequencing-based RAISING method, these mutants were successfully detected in the F0 generation. In F1 and F2 generations, droplet digital PCR analysis contributed to establishment of the strain, although a multicopy was falsely detected as one copy by the analysis in the F0 generation. We emphasize that the combination of these five methods allowed us to select promising F0 generations and establish the designed strain and that focusing only on positive evidence of knock-in can lead to erroneous selection of undesirable strains.

bioengineering↗