Tissue-specific and endogenous protein labeling with split fluorescent proteins.

Autor: Ligunas GD; Department of Molecular and Cell Biology, University of California, Merced, CA, USA; Quantitative and Systems Biology Graduate Group, University of California, Merced, CA, USA., Paniagua GF; Department of Molecular and Cell Biology, University of California, Merced, CA, USA., LaBelle J; Department of Molecular and Cell Biology, University of California, Merced, CA, USA; Quantitative and Systems Biology Graduate Group, University of California, Merced, CA, USA., Ramos-Martinez A; Department of Molecular and Cell Biology, University of California, Merced, CA, USA., Shen K; Department of Molecular and Cell Biology, University of California, Merced, CA, USA., Gerlt EH; Department of Molecular and Cell Biology, University of California, Merced, CA, USA., Aguilar K; Department of Molecular and Cell Biology, University of California, Merced, CA, USA., Nguyen N; Department of Molecular and Cell Biology, University of California, Merced, CA, USA., Materna SC; Department of Molecular and Cell Biology, University of California, Merced, CA, USA; Quantitative and Systems Biology Graduate Group, University of California, Merced, CA, USA; Health Sciences Research Institute, University of California, Merced, CA, USA., Woo S; Department of Molecular and Cell Biology, University of California, Merced, CA, USA; Quantitative and Systems Biology Graduate Group, University of California, Merced, CA, USA; Health Sciences Research Institute, University of California, Merced, CA, USA. Electronic address: swoo6@ucmerced.edu.
Jazyk: angličtina
Zdroj: Developmental biology [Dev Biol] 2024 Oct; Vol. 514, pp. 109-116. Date of Electronic Publication: 2024 Jun 21.
DOI: 10.1016/j.ydbio.2024.06.011
Abstrakt: The ability to label proteins by fusion with genetically encoded fluorescent proteins is a powerful tool for understanding dynamic biological processes. However, current approaches for expressing fluorescent protein fusions possess drawbacks, especially at the whole organism level. Expression by transgenesis risks potential overexpression artifacts while fluorescent protein insertion at endogenous loci is technically difficult and, more importantly, does not allow for tissue-specific study of broadly expressed proteins. To overcome these limitations, we have adopted the split fluorescent protein system mNeonGreen2 1-10/11 (split-mNG2) to achieve tissue-specific and endogenous protein labeling in zebrafish. In our approach, mNG2 1-10 is expressed under a tissue-specific promoter using standard transgenesis while mNG2 11 is inserted into protein-coding genes of interest using CRISPR/Cas-directed gene editing. Each mNG2 fragment on its own is not fluorescent, but when co-expressed the fragments self-assemble into a fluorescent complex. Here, we report successful use of split-mNG2 to achieve differential labeling of the cytoskeleton genes tubb4b and krt8 in various tissues. We also demonstrate that by anchoring the mNG2 1-10 component to specific cellular compartments, the split-mNG2 system can be used to manipulate protein localization. Our approach should be broadly useful for a wide range of applications.
(Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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