The <i>Caenorhabditis elegans</i> Transgenic Toolbox

Nance, Jeremy; Frøkjær-Jensen, Christian

doi:10.1534/genetics.119.301506

Cited by 140 publications

(108 citation statements)

References 272 publications

(328 reference statements)

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“…Similarly, researchers should also be aware that the tissue-specific promotors may also exhibit a low level of expression in other tissues, as was observed in this study (Radetskaya et al 2019). Transgenic approaches manipulating CRISPR-Cas9 activity in a tissue-specific manner (Shen et al 2014) or using conditional protein degradation methods may be more precise approaches for tissue-specific knockdown experiments in C. elegans (Nance and Frokjaer-Jensen 2019).…”

Section: Discussionmentioning

confidence: 66%

New strains for tissue-specific RNAi studies in Caenorhabditis elegans

Watts

Harrison

Omi

et al. 2018

Preprint

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RNA interference is a powerful tool for dissecting gene function. In Caenorhabditis elegans, ingestion of double stranded RNA causes strong, systemic knockdown of target genes. Further insight into gene function can be revealed by tissue-specific RNAi techniques. Currently available tissue-specific C. elegans strains rely on rescue of RNAi function in a desired tissue or cell in an otherwise RNAi deficient genetic background. In a classroom setting, we attempted to assess the contribution of specific tissues to polyunsaturated fatty acid (PUFA) synthesis using currently available tissue-specific RNAi strains. We discovered that rde-1 (ne219), a commonly used RNAi-resistant mutant strain, retains considerable RNAi capacity against RNAi directed at PUFA synthesis genes. Using GC/MS, we measured changes in the fatty acid products of the desaturase enzymes that synthesize PUFAs in a gene dosage sensitive manner.With this method, we tested a panel of previously described RNAi-deficient mutant strains, and found that almost all of them retained a certain degree of RNAi capacity. Importantly, we found that the before mentioned strain, rde-1 (ne219) and the reported germline only RNAi strain, rrf-1 (pk1417) are not appropriate genetic backgrounds for tissue-specific RNAi experiments.However, the knockout mutant rde-1 (ne300) was strongly resistant to dsRNA induced RNAi, and may be appropriate for construction of robust tissue-specific RNAi strains.

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Section: Discussionmentioning

confidence: 66%

New strains for tissue-specific RNAi studies in Caenorhabditis elegans

Watts

Harrison

Omi

et al. 2018

Preprint

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“…Plasmids were co-injected at concentration 17 ng/μL with marker pHS4 (lin-48pro::mRFP) at concentration 50 ng/μL. Integrated transgenes were generated using TMP/UV as described (Nance & Frøkjaer-Jensen, 2019) and plasmids generated for this study can be found in Table S2.…”

Section: Let-653 Transgenesmentioning

confidence: 99%

AC. elegansZona Pellucida domain protein functions via its ZPc domain

Cohen

Bermudez

Good

et al. 2020

Preprint

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Zona Pellucida domain (ZP) proteins are critical components of the body’s external-most protective layers, apical extracellular matrices (aECMs). Although their loss or dysfunction is associated with many diseases, it remains unclear how ZP proteins assemble in aECMs. Current models suggest that ZP proteins polymerize via their ZPn subdomains, while ZPc subdomains modulate ZPn behavior. Using the model organism C. elegans, we investigated the aECM assembly of one ZP protein, LET-653, which shapes several tubes. Contrary to prevailing models, we find that LET-653 localizes and functions via its ZPc domain. Furthermore, the ZPc domain is inhibited by the ZPn domain and cleavage of the LET-653 C-terminus relieves this inhibition. In vitro, the ZPc, but not ZPn, domain formed crystalline aggregates. These data offer a new model for ZP function whereby the ZPc domain is primarily responsible for matrix incorporation and tissue shaping.

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“…These methods have enabled important discoveries but can also lead to artifacts due to supraphysiological gene-expression levels and lack of endogenous regulatory control. In recent years, the repertoire of C. elegans transgenic tools has expanded [see (Nance and Frøkjaer-Jensen 2019) for review], particularly due to advances in CRISPR/Cas9 genome-editing technologies (Paix et al 2014;Dickinson and Goldstein 2016). CRISPR/Cas9 allows precise transgene insertion by homology-directed repair (HDR) and can be used to label an endogenous gene at its native locus with a fluorescent protein (Dokshin et al 2018;Farboud et al 2019;Vicencio et al 2019).…”

Section: Introductionmentioning

confidence: 99%

Split-wrmScarlet and split-sfGFP: Tools for faster, easier fluorescent labeling of endogenous proteins inCaenorhabditis elegans

Goudeau

Paw

Savy

et al. 2020

Preprint

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AbstractWe create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous C. elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest and can be detected wherever the large fragment is expressed and complemented. There is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet, and generate transgenic C. elegans lines to allow easy single-color labeling in muscles and dual-color labeling in somatic cells. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for an easy, cloning-free method for CRISPR/Cas9 editing.

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The Caenorhabditis elegans Transgenic Toolbox

Cited by 140 publications

References 272 publications

New strains for tissue-specific RNAi studies in Caenorhabditis elegans

New strains for tissue-specific RNAi studies in Caenorhabditis elegans

AC. elegansZona Pellucida domain protein functions via its ZPc domain

Split-wrmScarlet and split-sfGFP: Tools for faster, easier fluorescent labeling of endogenous proteins inCaenorhabditis elegans

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