Biology subjects

Sakuma, C.

Publications and source records attributed to Sakuma, C..

3 recordsLinked to original sources

bioRxiv2021

Whole-genome sequencing of Vero E6 (C1008) and comparative analysis of four Vero cell sublines

The Vero cell line is an immortalized cell line established from kidney epithelial cells of the African green monkey. A variety of sublines have been established from the original cell line, which display different characteristics. In this study, we determined the whole-genome sequence of Vero E6 (C1008) and performed comparative analysis among Vero JCRB 0111, Vero CCL-81, Vero 76 and Vero E6. Analysis of the copy number changes and loss of heterozygosity revealed that all sublines share a large deletion and loss of heterozygosity on chromosome 12, which harbors type I interferon and CDKN2 gene clusters. We identified a substantial number of genetic differences among the sublines including single nucleotide variants, indels, and copy number variations. The spectrum of single nucleotide variants indicated a close genetic relationship between Vero JCRB0111 and Vero CCL-81, and between Vero 76 and Vero E6, and a considerable genetic gap between the former two and the latter two lines. In contrast, we confirmed the pattern of genomic integration sites of simian endogenous retroviral sequences, which was consistent among the sublines. We identified subline-specific/enriched loss of function and missense variants, which potentially contribute to the differences in response to viral infection among the Vero sublines. In particular, we focused on Vero E6-specific/enriched variants and identified four genes (IL1RAP, TRIM25, RB1CC1, and ATG2A) that contained missense variants specific or enriched in Vero E6. In addition, we found that V739I variants of ACE2, which functions as the receptor for SARS-CoV-2, were heterozygous in Vero JCRB0111, Vero CCL-81, and Vero 76; however, Vero E6 contained the allele with isoleucine, resulting from the loss of one of the X chromosomes.

genomics↗

bioRxiv2021

Prospective isolation of mouse and human hematopoietic stem cells using Plexin domain containing 2

Numerous strategies exist to isolate hematopoietic stem cells (HSCs) using complex combinations of markers and flow cytometry. However, robust identification of HSCs using imaging techniques is substantially more challenging which has prompted the recent development of HSC reporter mice. To date, none of the molecules used in these reporters have been useful for human HSC identification. Here we report that PLXDC2 is a useful marker for both mouse and human HSCs. Using a green fluorescent protein (GFP) knock-in at the Plxdc2 locus in mice (hereafter denoted as Plxdc2-GFP), we showed that Plxdc2-GFP is highly expressed in HSCs with 1 in 2.8 Plxdc2-GFP+CD150+ cells giving long-term multi-lineage reconstitution in transplantation. Moreover, we developed a novel human PLXDC2 antibody and showed that human PLXDC2+ HSCs have stronger long-term multilineage reconstitution ability compared with PLXDC2- HSCs in a xenograft model. Thus, our study identifies PLXDC2 as a highly relevant molecule in HSC identification, potentially allowing greater purity and live in vivo tracking of these cells. SummaryTo date, few molecules are available for isolation of HSCs across species. The present study shows that PLXDC2 is a highly useful molecule for isolation of HSCs, which works across mouse and human.

cell biology↗

bioRxiv2021

The developmental hourglass model is applicable to the spinal cord

The developmental hourglass model predicts that embryonic morphology is most conserved at the mid-embryonic stage and diverges at the early and late stages. This model is generally considered by whole embryonic level. Here, we demonstrate that the hourglass model is also applicable to a reduced element, the spinal cord. In the middle of spinal cord development, dorsoventrally arrayed neuronal progenitor domains are established, which are conserved among vertebrates. We found that, by comparing the single-cell transcriptomes between mice and zebrafish, V3 interneurons, a subpopulation of the post-mitotic spinal neurons, display divergent molecular profiles. We also found non-conservation of cis-regulatory elements located around the progenitor fate determinants, indicating the rewiring of the upstream gene regulatory network. These results demonstrate that, despite the conservation of the progenitor domains, processes before and after the progenitor domain specification diverged. This study may help understand the molecular basis of the developmental hourglass model.

evolutionary biology↗