Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system

Heppert, Jennifer K.; Dickinson, Daniel J.; Pani, Ariel M.; Higgins, Christopher D.; Steward, Annette; Ahringer, Julie; Kuhn, Jeffrey R.; Goldstein, Bob

doi:10.1091/mbc.e16-01-0063

Cited by 122 publications

(111 citation statements)

References 52 publications

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“…However, the properties of these new fluorophores are generally characterized in bacteria or cell culture systems, and are not always retained in C. elegans cells or in specific subcellular compartments (Heppert et al 2016). …”

Section: Resultsmentioning

confidence: 99%

Reliable CRISPR/Cas9 Genome Engineering inCaenorhabditis elegansUsing a Single Efficient sgRNA and an Easily Recognizable Phenotype

Mouridi

Lecroisey

Tardy

et al. 2017

G3 Genes|Genomes|Genetics

106

110

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CRISPR/Cas9 genome engineering strategies allow the directed modification of the Caenorhabditis elegans genome to introduce point mutations, generate knock-out mutants, and insert coding sequences for epitope or fluorescent tags. Three practical aspects, however, complicate such experiments. First, the efficiency and specificity of single-guide RNAs (sgRNA) cannot be reliably predicted. Second, the detection of animals carrying genome edits can be challenging in the absence of clearly visible or selectable phenotypes. Third, the sgRNA target site must be inactivated after editing to avoid further double-strand break events. We describe here a strategy that addresses these complications by transplanting the protospacer of a highly efficient sgRNA into a gene of interest to render it amenable to genome engineering. This sgRNA targeting the dpy-10 gene generates genome edits at comparatively high frequency. We demonstrate that the transplanted protospacer is cleaved at the same time as the dpy-10 gene. Our strategy generates scarless genome edits because it no longer requires the introduction of mutations in endogenous sgRNA target sites. Modified progeny can be easily identified in the F1 generation, which drastically reduces the number of animals to be tested by PCR or phenotypic analysis. Using this strategy, we reliably generated precise deletion mutants, transcriptional reporters, and translational fusions with epitope tags and fluorescent reporter genes. In particular, we report here the first use of the new red fluorescent protein mScarlet in a multicellular organism. wrmScarlet, a C. elegans-optimized version, dramatically surpassed TagRFP-T by showing an eightfold increase in fluorescence in a direct comparison. KEYWORDS

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Section: Resultsmentioning

confidence: 99%

Reliable CRISPR/Cas9 Genome Engineering inCaenorhabditis elegansUsing a Single Efficient sgRNA and an Easily Recognizable Phenotype

Mouridi

Lecroisey

Tardy

et al. 2017

G3 Genes|Genomes|Genetics

106

110

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“…Since the pioneering use of green fluorescent protein (GFP) in C. elegans (Chalfie et al 1994), a number of fluorescent protein tags have been developed (for a comparative analysis of some of the currently available fluorescent probes, see Heppert et al 2016). Both intron insertion and codon optimization are important for robust fluorescent protein expression, especially in the germline (Green et al 2008).…”

Section: Engineering Cell Division Genesmentioning

confidence: 99%

“…In the last few years mNeonGreen, isolated from a lancelet, has emerged as a compelling alternative to green fluorescent protein (Shaner et al 2013). In our hands, mNeonGreen has improved the signal-to-noise ratio when fused to proteins such as cyclin B (CYB-1) (Kim et al 2017), although any potential improvement will depend on the target protein and/or the tissue/subcellular compartment in which it is expressed (Heppert et al 2016). …”

Section: Engineering Cell Division Genesmentioning

confidence: 99%

“…Red fluorescent proteins such as mCherry, mKate2 and TagRFP have also been adapted for use in C. elegans , although lower intensity and photostability of these proteins make them second choices for visualizing low abundance proteins (Heppert et al 2016). A novel mCherry variant called wrmScarlet (a codon-optimized version of mScarlet, Bindels et al 2017) was recently shown to be 6-fold brighter than TagRFP when fused to a muscle-expressed gene (El Mouridi et al 2017).…”

Section: Engineering Cell Division Genesmentioning

confidence: 99%

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Employing the one-cell C. elegans embryo to study cell division processes

Hattersley

Lara-González

Cheerambathur

et al. 2018

Methods in Cell Biology

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The one-cell Caenorhabditis elegans embryo offers many advantages for mechanistic analysis of cell division processes. Conservation of key genes and pathways involved in cell division makes findings in C. elegans broadly relevant. A key technical advantage of this system is the ability to penetrantly deplete essential gene products by RNA interference (RNAi) and replace them with wild-type or mutant versions expressed at endogenous levels from single copy RNAi-resistant transgene insertions. This ability to precisely perturb essential genes is complemented by the inherently highly reproducible nature of the zygotic division that facilitates development of quantitative imaging assays. Here, we detail approaches to generate targeted single copy transgene insertions that are RNAi-resistant, to engineer variants of individual genes employing transgene insertions as well as at the endogenous locus, and to in situ tag genes with fluorophores/purification tags. We also describe imaging assays and common image analysis tools employed to quantitatively monitor phenotypic effects of specific perturbations on meiotic and mitotic chromosome segregation, centrosome assembly/function, and cortical dynamics/cytokinesis.

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“…Here, we describe the currently and commercially available filters that can be used to rebuild these GFP-filter settings. In contrast to the single band FITC GFP filter set, the proposed triple band GFP filter set has a very narrow excitation bandwidth of 10 nm, which is right by the maximum peak for the S65C mutant GFP excitation (488 nm) that is commonly used in C. elegans (Boulin et al , 2006; Heppert et al , 2016). More importantly, the emission filter used here has a first pass-through (520/20 nm) for the light emitted close to the GFP emission peak (509 nm) and a second pass-through (595/40 nm) from the light around the autofluorescence emission, allowing the separation of GFP (visible in green) and autofluorescence (visible in yellow).…”

Section: Introductionmentioning

confidence: 99%

Overcoming Autofluorescence to Assess GFP Expression During Normal Physiology and Aging in Caenorhabditis elegans

Teuscher¹,

Ewald²

2018

BIO-PROTOCOL

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Green fluorescent protein (GFP) is widely used as a molecular tool to assess protein expression and localization. In C. elegans, the signal from weakly expressed GFP fusion proteins is masked by autofluorescence emitted from the intestinal lysosome-related gut granules. For instance, the GFP fluorescence from SKN-1 transcription factor fused to GFP is barely visible with common GFP (FITC) filter setups. Furthermore, this intestinal autofluorescence increases upon heat stress, oxidative stress (sodium azide), and during aging, thereby masking GFP expression even from proximal tissues. Here, we describe a triple band GFP filter setup that separates the GFP signal from autofluorescence, displaying GFP in green and autofluorescence in yellow. In addition, yellow fluorescent protein (YFP) remains distinguishable from both the yellowish autofluorescence and GFP with this triple band filter setup. Although some GFP intensity might be lost with the triple band GFP filter setup, the advantage is that no modification of currently used transgenic GFP lines is needed and these GFP filters are easy to install. Hence, by using this triple band GFP filter setup, the investigators can easily distinguish autofluorescence from GFP and YFP in their favorite transgenic C. elegans lines.

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Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system

Cited by 122 publications

References 52 publications

Reliable CRISPR/Cas9 Genome Engineering inCaenorhabditis elegansUsing a Single Efficient sgRNA and an Easily Recognizable Phenotype

Reliable CRISPR/Cas9 Genome Engineering inCaenorhabditis elegansUsing a Single Efficient sgRNA and an Easily Recognizable Phenotype

Employing the one-cell C. elegans embryo to study cell division processes

Overcoming Autofluorescence to Assess GFP Expression During Normal Physiology and Aging in Caenorhabditis elegans

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