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PreprintCited by 1Advanced Fluorescence Microscopy TechniquesCRISPR and Genetic EngineeringBiochemical and Structural Characterization

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Endogenous tagging using split mNeonGreen in human iPSCs for live imaging studies

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Husser, Mathieu C., Pham, Nhat P., Law, Chris, Araujo, Flavia R. B., Martin, Vincent J.J., Piekny, Alisa·2023·bioRxiv (Cold Spring Harbor Laboratory)
DOI 10.1101/2023.06.28.546942

Preprint — published as 10.7554/elife.92819 · Paper page

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Abstract

Endogenous tags have become invaluable tools to visualize and study native proteins in live cells. However, generating human cell lines carrying endogenous tags is difficult due to the low efficiency of homology-directed repair. Recently, an engineered split mNeonGreen protein was used to generate a large-scale endogenous tag library in HEK293 cells. Using split mNeonGreen for large-scale endogenous tagging in human iPSCs would open the door to studying protein function in healthy cells and across differentiated cell types. We engineered an iPS cell line to express the large fragment of the split mNeonGreen protein (mNG2 1-10 ) and showed that it enables fast and efficient endogenous tagging of proteins with the short fragment (mNG2 11 ). We also demonstrate that neural network-based image restoration enables live imaging studies of highly dynamic cellular processes such as cytokinesis in iPSCs. This work represents the first step towards a genome-wide endogenous tag library in human stem cells.

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Metadata

Authors
Husser, M. C., Pham, N. P., Law, C., Araujo, F. R. B., Martin, V. J. J., & Piekny, A.
Affiliations
Concordia University (CA)
Corresponding author
  • Martin, Vincent J.J.
  • Piekny, Alisa
ORCID
Venue
bioRxiv (Cold Spring Harbor Laboratory)
Publisher
openRxiv
Published
2023-06-28
DOI
10.1101/2023.06.28.546942
OpenAlex
W4382371052
Cited by
1
References
1
Citations by year
2025: 1
Type
posted content
Language
en
Open access
Licence
cc-by-nc-nd
Locations
bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
Funders
  • Concordia University
  • Natural Sciences and Engineering Research Council of Canada (04805, rgpin-2023-04805)
  • Fonds de recherche du Québec – Nature et technologies
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
Keywords
endogenous tagging, human induced pluripotent stem cells, live-cell imaging, homology-directed repair, cytokinesis, HEK293 cells

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