Endogenous tagging using split mNeonGreen in human iPSCs for live imaging studies
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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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Self-citationHusser, M. C., Ozugergin, I., Resta, T., Martin, V. J. J., & Piekny, A. J. (2022) · Open Biology · doi:10.1098/rsob.220247 · Cited by 11 · see citing works
Self-citationIwasaki, M., Kawahara, Y., Okubo, C., Yamakawa, T., Nakamura, M., Tabata, T., Nishi, Y., Narita, M., Ohta, A., Saito, H., et al. (2022) · iScience · doi:10.1016/j.isci.2022.104289 · Cited by 10 · see citing works
Efficient generation of endogenous protein reporters for mouse development
O’Hagan, D., Kruger, R. E., Gu, B., & Ralston, A. (2021) · Development · doi:10.1242/dev.197418 · Cited by 10 · see citing works
Diversity is the spice of life: An overview of how cytokinesis regulation varies with cell type
Ozugergin, I., & Piekny, A. J. (2022) · Frontiers in Cell and Developmental Biology · doi:10.3389/fcell.2022.1007614 · Cited by 7 · see citing works
Self-citation
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Yamashita, K., & Muramoto, T. (2025) · PLoS ONE · doi:10.1371/journal.pone.0326577 · Cited by 2 · see citing works
Lou, D., Kirrmaier, D., Krause, M., Li, S., Gubicza, K., Herbst, K., Meurer, M., Yerlici, V. T., Wagih, O., Steinmetz, L. M., et al. (2025) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.64898/2025.12.02.690677 · Cited by 1 · see citing works
- Authors
- Husser, M. C., Pham, N. P., Law, C., Araujo, F. R., Martin, V. J., & Piekny, A.
- Affiliations
- Concordia University (US)
- Concordia University (CA)
- Corresponding author
- Martin, Vincent JJ
- Piekny, Alisa
- Venue
- eLife
- Publisher
- eLife Sciences Publications, Ltd
- Published
- 2024-04-23
- Volume
- 12
- ISSN
- 2050-084X (ISSN-L)
- PMC
- PMC11037917
- OpenAlex
- W4388125678
- Cited by
- 2
- References
- 92
- Citations by year
- 2025: 2
- FWCI
- 0.24
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- eLife (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- 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, protein structure visualization, cytokinesis, HEK293 cells
- MeSH terms
- Cell Line, Humans, Induced Pluripotent Stem Cells, HEK293 Cells
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