Biology subjects

Wakimoto, A.

Publications and source records attributed to Wakimoto, A..

3 recordsLinked to original sources

bioRxiv2024

Intraplacental injection of human iPSC-derived PDX1+ pancreatic progenitors prolongs Pdx1-deficient mice survival

Interspecies chimeras comprising human tissues have potential for use in disease modeling and regenerative medicine. Here, we successfully transplanted human induced pluripotent stem cell (iPSC)-derived PDX1+ pancreatic progenitor cells into Pdx1-deficient mouse embryos via intraplacental injection. The engrafted human cells predominantly localized to the duodenum, produced insulin, and extended the lifespan of Pdx1-/- mice by up to 10 days after birth. Transcriptomic analyses confirmed human pancreatic gene expression in human cells engrafted into the mouse duodenum. Our findings demonstrated the feasibility of generating interspecies chimeras with functional human pancreatic cells through in utero transplantation of lineage-committed progenitors. This approach circumvents developmental barriers while minimizing ethical concerns associated with PSCs. However, the incomplete rescue of the Pdx1-/- phenotype highlights the need for further research to enhance human cell engraftment and tissue integration. Overall, this study provides a foundation for developing human-animal chimera models for studying human development and regenerative therapies.

cell biology↗

bioRxiv2020

DAJIN-assisted multiplex genotyping to validate the outcomes of CRISPR-Cas-based genome editing

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

genomics↗

bioRxiv2018

Homologous expression and characterization of gassericin T and gassericin S, a novel class IIb bacteriocin produced by Lactobacillus gasseri LA327

Lactobacillus gasseri LA327 isolated from the large intestine tissue in humans is a bacteriocinogenic strain and is predicted to produce two kinds of class IIb bacteriocins, i.e. gassericin T (GT) and acidocin LF221A (Acd LF221A). In this study, DNA sequencing of the genes for GT and Acd LF221A on Lb. gasseri LA327 revealed that the amino acid sequences for GT completely corresponded with those of gat except for GatK (histidine kinase). However, those for the Acd LF221A had analogues which differed in at least one amino acid residue to be a putative class IIb bacteriocin designated as gassericin S (GS). By deletion test of GT structural genes (gatAX), the LA327 strain retained the bacteriocin activity, and the LA327 mutant strain lacking the ABC-type transporter gene (gatT) completely lost the bacteriocin activity. This indicates that LA327 strain is a GS producer, and GS production is performed via gat with the inclusion of gatT. Homologous expression using deletion mutants for GS and GT containing each single peptide elucidated that GS (GasAX) and GT (GatAX) showed synergistic activity as class IIb bacteriocins, respectively, and no synergistic activity was observed between each peptide of GS and GT. The molecular mass of GS was estimated to be theoretical ca. 5,400 Da by in situ activity assay after SDS-PAGE, clarifying that GS was actually expressed as an active class IIb bacteriocin. Furthermore, stability of GS expressed against pH, heat and protease was determined.\n\nImportanceWe determined the complete DNA sequence for GS, a novel class IIb bacteriocin of Lb. gasseri, and succeeded to express GS as active bacteriocins. Our results clarified the interaction of each class IIb component peptide for GT in addition to GS via construction of homologous mutants which were not dependent on the purification. These data may demonstrate the characteristics of class IIb bacteriocins for Lb. gasseri.

microbiology↗