Engineering and characterisation of AAVS1 FRT cell lines for inducible and dose-dependent gene expression
- 1Division of Cell and Molecular Biology, The Institute of Cancer Research, London, UK, Department of Cell and Molecular Biology, St. Jude Children?s Research Hos
Abstract
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.
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BibTeX
@misc{malumbres2026engineering,
title = {{Engineering and characterisation of AAVS1 FRT cell lines for inducible and dose-dependent gene expression}},
author = {M. Portillo Malumbres and L. Ellingboe and A. Roberti and O. Nashchekina and F. J. Sialana and J. Pines and J. S. Choudhary and J. Mansfeld and N. E. Davey},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.29.762215},
url = {https://www.biorxiv.org/content/10.64898/2026.10.29.762215v1},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - Engineering and characterisation of AAVS1 FRT cell lines for inducible and dose-dependent gene expression
AU - M. Portillo Malumbres
AU - L. Ellingboe
AU - A. Roberti
AU - O. Nashchekina
AU - F. J. Sialana
AU - J. Pines
AU - J. S. Choudhary
AU - J. Mansfeld
AU - N. E. Davey
PY - 2026
DA - 2026/10/29
PB - bioRxiv
DO - 10.64898/2026.10.29.762215
UR - https://www.biorxiv.org/content/10.64898/2026.10.29.762215v1
AB - 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.
ER -