A toolkit for GFP-mediated tissue-specific protein degradation in C. elegans
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Abstract
Proteins that are essential for embryo production, cell division and early embryonic events are frequently reused later in embryogenesis, during organismal development or in the adult. Examining protein function across these different biological contexts requires tissue-specific perturbation. Here, we describe a method that uses expression of a fusion between a GFP-targeting nanobody and a SOCS-box containing ubiquitin ligase adaptor to target GFP-tagged proteins for degradation. When combined with endogenous locus GFP tagging by CRISPR-Cas9 or with rescue of a null mutant with a GFP fusion, this approach enables routine and efficient tissue-specific protein ablation. We show that this approach works in multiple tissues – the epidermis, intestine, body wall muscle, ciliated sensory neurons and touch receptor neurons – where it recapitulates expected loss-of-function mutant phenotypes. The transgene toolkit and the strain set described here will complement existing approaches to enable routine analysis of the tissue-specific roles of C. elegans proteins.
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The ZIF1-GFPnb polypeptide recruits and degrades GFP-tagged knockin POIs in vivo.
“In combination with CRISPR/Cas9 gene editing technology, it was demonstrated that the ZIF1–GFPnb polypeptide recruits and degrades GFP-tagged knockin POIs in vivo (Fig. 3b) [50].”
From Advances in targeted degradation of endogenous proteins (2019) · cites this paper as [50]
deGradFP, a fusion of an anti-GFP nanobody with the F-box domain from Drosophila melanogaster Slmb, efficiently degrades GFP-tagged proteins in D. melanogaster.
“One such approach, named deGradFP, utilized a fusion of an anti-GFP nanobody with the F-box domain from the Drosophila melanogaster Slmb protein, which is a substrate receptor component of the SCF E3 ligase complex, and demonstrated efficient degradation of GFP-tagged proteins in D. melanogaster (Fig. 3b) [50, 51].”
From Advances in targeted degradation of endogenous proteins (2019) · cites this paper as [50]
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References 32 · Cited by 119
- Authors
- Wang, S., Tang, N. H., Lara-Gonzalez, P., Zhao, Z., Cheerambathur, D. K., Prevo, B., Chisholm, A. D., Desai, A., & Oegema, K.
- Affiliations
- University of California San Diego (US)
- Ludwig Institute for Cancer Research 1 , Department of Cellular and Molecular Medicine , , La Jolla, CA 92093 , USA
- Corresponding author
- Oegema, Karen
- ORCID
- Venue
- Development
- Publisher
- The Company of Biologists
- Published
- 2017-07-15
- Volume
- 144
- Issue
- 14
- Pages
- 2694-2701
- ISSN
- 0950-1991 (ISSN-L)
- 1477-9129
- PMC
- PMC5536931
- OpenAlex
- W2625678278
- Cited by
- 119
- References
- 32
- Citations by year
- 2026: 4 · 2025: 13 · 2024: 11 · 2023: 14 · 2022: 20 · 2021: 17 · 2020: 16 · 2019: 14 · 2018: 10
- FWCI
- 4.47
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Locations
- Development (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- HighWire Press Open Archive (submittedVersion)
- Funders
- National Institutes of Health (GM074207, NS093588)
- Ludwig Institute for Cancer Research
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Developmental Biology and Gene Regulation
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Aging › Genetics, Aging, and Longevity in Model Organisms
- Keywords
- CRISPR/Cas9, GFP tagging, epidermis, intestine, body wall muscle, touch receptor neurons, Caenorhabditis elegans
- MeSH terms
- Animals, Axons, Carrier Proteins, Genetic Techniques, Mutation, Recombinant Fusion Proteins, Tissue Distribution, Caenorhabditis elegans, Genes, Helminth, MAP Kinase Kinase Kinases, Caenorhabditis elegans Proteins, Animals, Genetically Modified, Green Fluorescent Proteins, Proteolysis, Single-Domain Antibodies
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