scholarly journals Robust genome editing with short single-stranded and long, partially single-stranded DNA donors in C. elegans

2018 ◽  
Author(s):  
Gregoriy A. Dokshin ◽  
Krishna S. Ghanta ◽  
Katherine M. Piscopo ◽  
Craig C. Mello
Keyword(s):  
Green Fluorescent Protein ◽  
Cost Effectiveness ◽  
Genome Editing ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
C Elegans ◽  
Multiple Loci ◽  
Green Fluorescent ◽  
Fluorescent Protein Tags ◽  
Protein Tags

AbstractCRISPR-based genome editing using ribonucleoprotein (RNP) complexes and synthetic single stranded oligodeoxynucleotide (ssODN) donors can be highly effective. However, reproducibility can vary, and precise, targeted integration of longer constructs – such as green fluorescent protein (GFP) tags remains challenging in many systems. Here we describe a streamlined and optimized editing protocol for the nematode C. elegans. We demonstrate its efficacy, flexibility, and cost-effectiveness by affinity-tagging all twelve of the Worm-specific Argonaute (WAGO) proteins in C. elegans using ssODN donors. In addition, we describe a novel PCR-based partially single-stranded “hybrid” donor design that yields high efficiency editing with large (kilobase-scale) constructs. We use these hybrid donors to introduce fluorescent protein tags into multiple loci achieving editing efficiencies that approach those previously obtained only with much shorter ssODN donors. The principals and strategies described here are likely to translate to other systems and should allow researchers to reproducibly and efficiently obtain both long and short precision genome edits.

scholarly journals Green fluorescent protein as a scaffold for high efficiency production of functional bacteriotoxic proteins in Escherichia coli

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Nagasundarapandian Soundrarajan ◽  
Hye-sun Cho ◽  
Byeongyong Ahn ◽  
Minkyung Choi ◽  
Le Minh Thong ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
Green Fluorescent

A novel WD40 protein, CHE-2, acts cell-autonomously in the formation of C. elegans sensory cilia

Development ◽  
1999 ◽  
Vol 126 (21) ◽  
pp. 4839-4848 ◽  
Author(s):  
M. Fujiwara ◽  
T. Ishihara ◽  
I. Katsura
Keyword(s):  
Green Fluorescent Protein ◽  
Sensory Neurons ◽  
Fluorescent Protein ◽  
Interaction Analysis ◽  
C Elegans ◽  
Protein Protein Interaction ◽  
Amino Terminal ◽  
Wd40 Domain ◽  
Green Fluorescent ◽  
Almost All

To elucidate the mechanism of sensory cilium formation, we analyzed mutants in the Caenorhabditis elegans che-2 gene. These mutants have extremely short cilia with an abnormal posterior projection, and show defects in behaviors that are mediated by ciliated sensory neurons. The che-2 gene encodes a new member of the WD40 protein family, suggesting that it acts in protein-protein interaction. Analysis of mutation sites showed that both the amino-terminal WD40 repeats and the carboxyl-terminal non-WD40 domain are necessary for the CHE-2 function. CHE-2-tagged green fluorescent protein is localized at the cilia of almost all the ciliated sensory neurons. Expression of che-2 in a subset of sensory neurons of a che-2 mutant by using a heterologous promoter resulted in restoration of the functions and cilium morphology of only the che-2-expressing neurons. Thus, che-2 acts cell-autonomously. This technique can be used in the future for determining the function of each type of che-2-expressing sensory neuron. Using green fluorescent protein, we found that the extension of cilia in wild-type animals took place at the late embryonic stage, whereas the cilia of che-2 mutant animals remained always short during development. Hence, the abnormal posterior projection is due to the inability of cilia to extend, rather than degeneration of cilia once correctly formed. Expression of che-2 in a che-2 mutant under a heat shock promoter showed that the extension of cilia, surprisingly, can occur even at the adult stage, and that such cilia can function apparently normally in behavior.

scholarly journals eat-5 and unc-7 represent a multigene family in Caenorhabditis elegans involved in cell-cell coupling.

1996 ◽  
Vol 134 (2) ◽  
pp. 537-548 ◽  
Author(s):  
T A Starich ◽  
R Y Lee ◽  
C Panzarella ◽  
L Avery ◽  
J E Shaw
Keyword(s):  
Green Fluorescent Protein ◽  
Caenorhabditis Elegans ◽  
Gene Family ◽  
Plasma Membranes ◽  
Fluorescent Protein ◽  
Amino Acid Sequences ◽  
C Elegans ◽  
Pharyngeal Muscles ◽  
Green Fluorescent ◽  
Posterior Pharynx

The Drosophila melanogaster genes Passover and l(1)ogre and the Caenorhabditis elegans gene unc-7 define a gene family whose function is not known. We have isolated and characterized the C. elegans gene eat-5, which is required for synchronized pharyngeal muscle contractions, and find that it is a new member of this family. Simultaneous electrical and video recordings reveal that in eat-5 mutants, action potentials of muscles in the anterior and posterior pharynx are unsynchronized. Injection of carboxyfluorescein into muscles of the posterior pharynx demonstrates that all pharyngeal muscles are dye-coupled in wild-type animals; in eat-5 mutants, however, muscles of the anterior pharynx are no longer dye-coupled to posterior pharyngeal muscles. We show that a gene fusion of eat-5 to the green fluorescent protein is expressed in pharyngeal muscles. unc-7 and eat-5 are two of at least sixteen members of this family in C. elegans as determined by database searches and PCR-based screens. The amino acid sequences of five of these members in C. elegans have been deduced from cDNA sequences. Polypeptides of the family are predicted to have four transmembrane domains with cytoplasmic amino and carboxyl termini. We have constructed fusions of one of these polypeptides with beta-galactosidase and with green fluorescent protein. The fusion proteins appear to be localized in a punctate pattern at or near plasma membranes. We speculate that this gene family is required for the formation of gap junctions.

Immuno-EM Localization of GFP-tagged Yolk Proteins in C. Elegans Using Microwave Fixation

2001 ◽  
Vol 49 (8) ◽  
pp. 949-956 ◽  
Author(s):  
Marie-Christine Paupard ◽  
Agnes Miller ◽  
Barth Grant ◽  
David Hirsh ◽  
David H. Hall
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Cell Types ◽  
Protein Fusion ◽  
C Elegans ◽  
High Resolution Analysis ◽  
Resolution Analysis ◽  
Microwave Fixation ◽  
Green Fluorescent ◽  
Transgenic Strains

Because of the presence of a low-permeability cuticle covering the animal, fixation of C. elegans tissue for immunoelectron microscopy has proved very difficult. Here we applied a microwave fixation protocol to improve penetration of fixatives before postembedding immunogold labeling. Using this technique, we were able to successfully localize several components of yolk (YP170) trafficking in both wild-type and transgenic strains expressing a vitellogenin::green fluorescent protein fusion (YP170::GFP). Green fluorescent protein (GFP) and its variants are commonly used as markers to localize proteins in transgenic C. elegans using fluorescence microscopy. We have developed a robust method to localize GFP at the EM level. This procedure is applicable to the characterization of transgenic strains in which GFP is used to mark particular proteins or cell types and will undoubtedly be very useful for high-resolution analysis of marked structures. (J Histochem Cytochem 49:949–956, 2001)

scholarly journals Proteins pinpoint double strand breaks

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Michael M Cox
Keyword(s):  
Dna Damage ◽  
Green Fluorescent Protein ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Green Fluorescent ◽  
Important Form ◽  
Living Bacteria

Combining green fluorescent protein with a protein that only binds to double strand breaks in DNA allows these breaks—which are an important form of DNA damage—to be detected with high efficiency in living bacteria.

scholarly journals Comparative assessment of fluorescent proteins forin vivoimaging in an animal model system

2016 ◽  
Author(s):  
Jennifer K Heppert ◽  
Daniel J Dickinson ◽  
Ariel M Pani ◽  
Christopher D Higgins ◽  
Annette Steward ◽  
...  
Keyword(s):  
Animal Model ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Model System ◽  
C Elegans ◽  
Red Fluorescent Proteins ◽  
Fluorescent Protein Tags ◽  
Protein Tags

Fluorescent protein tags are fundamental tools used to visualize gene products and analyze their dynamicsin vivo. Recent advances in genome editing have enabled precise insertion of fluorescent protein tags into the genomes of diverse organisms. These advances expand the potential ofin vivoimaging experiments, and they facilitate experimentation with new, bright, photostable fluorescent proteins. Most quantitative comparisons of the brightness and photostability of different fluorescent proteins have been madein vitro, removed from biological variables that govern their performance in cells or organisms. To address the gap we quantitatively assessed fluorescent protein propertiesin vivoin an animal model system. We generated transgenicC. elegansstrains expressing green, yellow, or red fluorescent proteins in embryos, and we imaged embryos expressing different fluorescent proteins under the same conditions for direct comparison. We found that mNeonGreen was not brightin vivoas predicted based onin vitrodata, but that mNeonGreen is a better tag than GFP for specific kinds of experiments, and we report on optimal red fluorescent proteins. These results identify ideal fluorescent proteins for imagingin vivoinC. elegansembryos, and they suggest good candidate fluorescent proteins to test in other animal model systems.

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