Biology subjects

Nashchekina, O.

Publications and source records attributed to Nashchekina, O..

2 recordsLinked to original sources

bioRxiv2026

Engineering and characterisation of AAVS1 FRT cell lines for inducible and dose-dependent gene expression

Investigating biological function with high reproducibility requires the generation of stable cell lines that offer precise and inducible transgene expression. However, traditional methods often suffer from the limitations of random genomic integration, including unpredictable expression levels, leaky basal activity, and disruption of endogenous loci. To address these challenges, we engineered a versatile RPE1 cell line by integrating a TetOn3G-FRT cassette into the AAVS1 safe-harbour locus using CRISPR/Cas9(D10A) nickase. This platform utilises the Flp-FRT recombination system to facilitate rapid, site-specific "plug-and-play" insertion of transgenes, combined with the Tet-On 3G system for tight, dose-dependent expression control. The resulting RPE1 AAVS1 FRT8 line enables highly consistent transgene induction across independent clones, behaving as a monoclonal population. We highlight the importance of rigorous cell line characterisation through a multi-omics approach. Whole genome sequencing identified precise heterozygous integration at the target locus and de novo genomic duplications on other chromosomes. By integrating WGS with high-resolution proteomics and cell cycle analysis, we confirmed that these alterations did not significantly perturb cellular homeostasis or phenotypic fitness. This work establishes a robust and reliable platform for the efficient generation of inducible AAVS1 FRT stable cell lines, facilitating reproducible research in modern molecular biology.

bioengineering↗

bioRxiv2025

Decoding human B cell ontogeny in prenatal and adult bone marrow and in vitro models via single-cell multiomics

In humans, the bone marrow becomes the primary site for B lymphopoiesis during the second trimester of pregnancy and continues throughout life. Prenatal and adult B cell progenitors play distinct roles in the aetiology and pathology of paediatric and adult hematopoietic malignancies, though the molecular drivers of these differences remain unclear. Here, we created a comprehensive multiomics atlas of over 500k cells covering immune and stromal compartment from prenatal and adult bone marrow, enabling high-resolution analysis of the cell-intrinsic and cell-extrinsic processes that modulate prenatal and adult B lymphopoiesis. Even though B cells follow broadly similar developmental trajectories, we identify a novel postnatal lateCLP subset equivalent to prenatal preProB cells, and uncover prenatal cells carry several signatures characteristic of leukemias, including enhanced proliferation, higher RAG1/RAG2 activity, extrinsic B cell signals such as IL7, and lower retention signals in the bone marrow. We also developed and characterised, at both cellular and molecular levels, a new experimental framework for generating B cell precursors from human induced pluripotent stem cells (hiPSCs), and show that it faithfully recapitulates key stages of B cell differentiation. Together, our single-cell multiomics atlas of B lymphopoiesis in vivo and in vitro offers detailed insights into the unique molecular features of prenatal and adult B cell lymphopoiesis, and serves as a powerful resource for investigating the early events that contribute to haematological disorders.

immunology↗