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PreprintCited by 0Advanced Fluorescence Microscopy TechniquesCRISPR and Genetic EngineeringPluripotent Stem Cells Research

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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·2024·eLife
DOI 10.7554/elife.92819.2

Preprint — published as 10.7554/elife.92819.3 · Paper page

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Abstract

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 (mNG21-10) and showed that it enables fast and efficient endogenous tagging of proteins with the short fragment (mNG211). 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 (US)
  • Concordia University (CA)
ORCID
Venue
eLife
Publisher
eLife Sciences Publications, Ltd
Published
2024-03-26
ISSN
2050-084X (ISSN-L)
DOI
10.7554/elife.92819.2
OpenAlex
W4393200594
Cited by
0
References
92
Type
posted content
Language
en
Open access
Licence
Locations
eLife (acceptedVersion, cc-by)
Funders
  • Fonds de recherche du Québec – Nature et technologies (Doctoral Training Scholarship B2X)
  • Concordia University (SynBioApps scholarship)
  • National Research Council Canada (Disruptive Technology Solutions for Cell and Gene Therapy Challenge)
  • Natural Sciences and Engineering Research Council of Canada (Discovery Grant, RGPIN-2023-04805)
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 › Pluripotent Stem Cells Research
Keywords
endogenous tagging, human induced pluripotent stem cells, live-cell imaging, homology-directed repair, cytokinesis, protein structure visualization

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