scholarly journals Extrachromosomal markers for bacteriophage phiC31 recombinase actions on C. elegans genome.

2021 ◽  
Author(s):  
Wadim J Kapulkin ◽  
Andrei Pozniakovsky
Keyword(s):  
Proof Of Concept ◽  
Phic31 Integrase ◽  
C Elegans ◽  
Experimental Implementation ◽  
Split System

We report on the experimental implementation of the transgenic phiC31 recombinase C. elegans intron-split system. The three-component plasmid-based phiC31 recombinase system consists of i) two intron-split segments of the C. briggsae-unc-119 gene, and ii) the plasmid that provides phiC31 recombinase activity. Described results constitute the proof-of-concept assay for the implementation of bacteriophage phiC31 integrase in C. elegans.

scholarly journals Identification of high-efficiency 3′GG gRNA motifs in indexed FASTA files with ngg2

2015 ◽  
Vol 1 ◽  
pp. e33 ◽  
Author(s):  
Elisha D. Roberson
Keyword(s):  
High Efficiency ◽  
Homo Sapiens ◽  
Model Organisms ◽  
Proof Of Concept ◽  
Protein Coding ◽  
C Elegans ◽  
Protein Coding Genes ◽  
Starting Point ◽  
Command Line Tool ◽  
Reference Genomes

CRISPR/Cas9 is emerging as one of the most-used methods of genome modification in organisms ranging from bacteria to human cells. However, the efficiency of editing varies tremendously site-to-site. A recent report identified a novel motif, called the 3′GG motif, which substantially increases the efficiency of editing at all sites tested inC. elegans. Furthermore, they highlighted that previously published gRNAs with high editing efficiency also had this motif. I designed a Python command-line tool, ngg2, to identify 3′GG gRNA sites from indexed FASTA files. As a proof-of-concept, I screened for these motifs in six model genomes:Saccharomyces cerevisiae,Caenorhabditis elegans,Drosophila melanogaster,Danio rerio,Mus musculus, andHomo sapiens. I also scanned the genomes of pig (Sus scrofa) and African elephant (Loxodonta africana) to demonstrate the utility in non-model organisms. I identified more than 60 million single match 3′GG motifs in these genomes. Greater than 61% of all protein coding genes in the reference genomes had at least one unique 3′GG gRNA site overlapping an exon. In particular, more than 96% of mouse and 93% of human protein coding genes have at least one unique, overlapping 3′GG gRNA. These identified sites can be used as a starting point in gRNA selection, and the ngg2 tool provides an important ability to identify 3′GG editing sites in any species with an available genome sequence.

scholarly journals Caenorhabditis elegans Infrared-Based Motility Assay Identified New Hits for Nematicide Drug Development

2019 ◽  
Vol 6 (1) ◽  
pp. 29 ◽  
Author(s):  
Gastón Risi ◽  
Elena Aguilera ◽  
Enrique Ladós ◽  
Gonzalo Suárez ◽  
Inés Carrera ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Model Organism ◽  
Proof Of Concept ◽  
Free Living ◽  
C Elegans ◽  
Nematode Parasites ◽  
Automated Assay ◽  
Free Living Nematode ◽  
Vertebrate Model ◽  
Electronegative Atom

Nematode parasites have a profound impact on humankind, infecting nearly one-quarter of the world’s population, as well as livestock. There is a pressing need for discovering nematicides due to the spread of resistance to currently used drugs. The free-living nematode Caenorhabditis elegans is a formidable experimentally tractable model organism that offers key advantages in accelerating nematicide discovery. We report the screening of drug-like libraries using an overnight high-throughput C. elegans assay, based on an automated infrared motility reader. As a proof of concept, we screened the “Pathogen Box” library, and identical results to a previous screen using Haemonchus contortus were obtained. We then screened an in-house library containing a diversity of compound families. Most active compounds had a conjugation of an unsaturation with an electronegative atom (N, O, or S) and an aromatic ring. Importantly, we identified symmetric arylidene ketones and aryl hydrazine derivatives as novel nematicides. Furthermore, one of these compounds, (1E,2E)-1,2-bis(thiophen-3-ylmethylene)hydrazine, was active as a nematicide at 25 µm, but innocuous to the vertebrate model zebrafish at 50 µm. Our results identified novel nematicidal scaffolds and illustrate the value of C. elegans in accelerating nematicide discovery using a nonlabor-intensive automated assay that provides a simple overnight readout.

scholarly journals Identification of high-efficiency 3’GG gRNA motifs in indexed FASTA files with ngg2

2015 ◽  
Author(s):  
Elisha D Roberson
Keyword(s):  
High Efficiency ◽  
Homo Sapiens ◽  
Model Organisms ◽  
Proof Of Concept ◽  
Protein Coding ◽  
C Elegans ◽  
Protein Coding Genes ◽  
Starting Point ◽  
Command Line Tool ◽  
Reference Genomes

CRISPR/Cas9 is emerging as one of the most-used methods of genome modification in organisms ranging from bacteria to human cells. However, the efficiency of editing varies tremendously site-to-site. A recent report identified a novel motif, called the 3’GG motif, which substantially increases the efficiency of editing at all sites tested in C. elegans. Furthermore, they highlighted that previously published gRNAs with high editing efficiency also had this motif. I designed a python command-line tool, ngg2, to identify 3’GG gRNA sites from indexed FASTA files. As a proof-of-concept, I screened for these motifs in six model genomes: Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Mus musculus, and Homo sapiens. I also scanned the genomes of pig (Sus scrofa) and African elephant (Loxodonta africana) to demonstrate the utility in non-model organisms. I identified more than 60 million single match 3’GG motifs in these genomes. Greater than 61% of all protein coding genes in the reference genomes had at least one unique 3’GG gRNA site overlapping an exon. In particular, more than 96% of mouse and 93% of human protein coding genes have at least one unique, overlapping 3’GG gRNA. These identified sites can be used as a starting point in gRNA selection, and the ngg2 tool provides an important ability to identify 3'GG editing sites in any species with an available genome sequence.

scholarly journals phiC31 integrase for recombination mediated single copy insertion and genome manipulation in C. elegans

Genetics ◽  
2021 ◽  
Author(s):  
Fang-Jung Yang ◽  
Chiao-Nung Chen ◽  
Tiffany Chang ◽  
Ting-Wei Cheng ◽  
Ni-Chen Chang ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Integration Method ◽  
Single Copy ◽  
Large Set ◽  
Reporter System ◽  
Phic31 Integrase ◽  
C Elegans ◽  
Cassette Exchange ◽  
Post Transcriptional Regulation ◽  
Genome Manipulation

Abstract C. elegans benefits from a large set of tools for genome manipulation. Yet, the precise single-copy insertion of very large DNA constructs (>10 kb) and the generation of inversions are still challenging. Here, we adapted the phiC31 integrase system for C. elegans. We generated an integrated phiC31 integrase expressing strain flanked by attP sites that serves as a landing pad for integration of transgenes by recombination mediated cassette exchange (RCME). This strain is unc-119(-) so RMCE integrants can be produced simply by injection of a plasmid carrying attB sites flanking unc-119(+) and the gene(s) of interest. Additionally, phiC31 integrase is removed concomitantly with integration, eliminating the need to outcross away the integrase. Integrations were obtained for insert sizes up to ∼33.4 kb. Taking advantage of this integration method we establish a dual color fluorescent operon reporter system able to study post-transcriptional regulation of mRNA. Last, we show that large chromosomal segments can be inverted using phiC31 integrase. Thus, the phiC31 integrase system should be a useful addition to the C. elegans toolkit.

scholarly journals Identification of high-efficiency 3’GG gRNA motifs in indexed FASTA files with ngg2

2015 ◽  
Author(s):  
Elisha D Roberson
Keyword(s):  
High Efficiency ◽  
Homo Sapiens ◽  
Model Organisms ◽  
Proof Of Concept ◽  
Protein Coding ◽  
C Elegans ◽  
Protein Coding Genes ◽  
Starting Point ◽  
Command Line Tool ◽  
Reference Genomes

CRISPR/Cas9 is emerging as one of the most-used methods of genome modification in organisms ranging from bacteria to human cells. However, the efficiency of editing varies tremendously site-to-site. A recent report identified a novel motif, called the 3’GG motif, which substantially increases the efficiency of editing at all sites tested in C. elegans. Furthermore, they highlighted that previously published gRNAs with high editing efficiency also had this motif. I designed a python command-line tool, ngg2, to identify 3’GG gRNA sites from indexed FASTA files. As a proof-of-concept, I screened for these motifs in six model genomes: Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Mus musculus, and Homo sapiens. I also scanned the genomes of pig (Sus scrofa) and African elephant (Loxodonta africana) to demonstrate the utility in non-model organisms. I identified more than 60 million single match 3’GG motifs in these genomes. Greater than 61% of all protein coding genes in the reference genomes had at least one unique 3’GG gRNA site overlapping an exon. In particular, more than 96% of mouse and 93% of human protein coding genes have at least one unique, overlapping 3’GG gRNA. These identified sites can be used as a starting point in gRNA selection, and the ngg2 tool provides an important ability to identify 3'GG editing sites in any species with an available genome sequence.

scholarly journals phiC31 integrase for recombination mediated single copy insertion and genome manipulation in C. elegans

2020 ◽  
Author(s):  
Fang-Jung Yang ◽  
Chiao-Nung Chen ◽  
Tiffany Chang ◽  
Ting-Wei Cheng ◽  
Ni-Chen Chang ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Integration Method ◽  
Single Copy ◽  
Large Set ◽  
Reporter System ◽  
Phic31 Integrase ◽  
C Elegans ◽  
Cassette Exchange ◽  
Post Transcriptional Regulation ◽  
Genome Manipulation

AbstractC. elegans benefits from a large set of tools for genome manipulation. Yet, the insertion of large DNA constructs and the generation of inversions is still challenging. Here, we adapted the phiC31 integrase system for C. elegans. We generated an integrated phiC31 integrase expressing strain flanked by attP sites that also serves as a landing pad for integration of transgenes by recombination mediated cassette exchange (RCME). This strain is unc-119(-) so RMCE integrants can be produced simply by injection of a plasmid carrying attB sites flanking unc-119(+) and the gene(s) of interest. Additionally, phiC31 integrase is removed concomitantly with integration, eliminating the need to outcross away the integrase. Integrations are relatively easy to obtain for insert sizes up to ~15 kb. Taking advantage of this integration method we establish a dual color fluorescent operon reporter system to study post-transcriptional regulation of mRNA. Last we show that large chromosomal segments can be inverted using phiC31 integrase. Thus the phiC31 integrase system should be a useful addition to the C. elegans toolkit.

Direct measurement of electron-diffraction-pattern intensities using an energy loss spectrometer

1989 ◽  
Vol 47 ◽  
pp. 668-669
Author(s):  
A. G. Jackson ◽  
M. Rowe
Keyword(s):  
Thermal Effects ◽  
Dynamic Range ◽  
Scattering Amplitude ◽  
Dynamical Theory ◽  
Site Symmetry ◽  
Proof Of Concept ◽  
Parallel Acquisition ◽  
Kinematic Approximation ◽  
Speed Up ◽  
Structure Calculations

Diffraction intensities from intermetallic compounds are, in the kinematic approximation, proportional to the scattering amplitude from the element doing the scattering. More detailed calculations have shown that site symmetry and occupation by various atom species also affects the intensity in a diffracted beam. [1] Hence, by measuring the intensities of beams, or their ratios, the occupancy can be estimated. Measurement of the intensity values also allows structure calculations to be made to determine the spatial distribution of the potentials doing the scattering. Thermal effects are also present as a background contribution. Inelastic effects such as loss or absorption/excitation complicate the intensity behavior, and dynamical theory is required to estimate the intensity value.The dynamic range of currents in diffracted beams can be 104or 105:1. Hence, detection of such information requires a means for collecting the intensity over a signal-to-noise range beyond that obtainable with a single film plate, which has a S/N of about 103:1. Although such a collection system is not available currently, a simple system consisting of instrumentation on an existing STEM can be used as a proof of concept which has a S/N of about 255:1, limited by the 8 bit pixel attributes used in the electronics. Use of 24 bit pixel attributes would easily allowthe desired noise range to be attained in the processing instrumentation. The S/N of the scintillator used by the photoelectron sensor is about 106 to 1, well beyond the S/N goal. The trade-off that must be made is the time for acquiring the signal, since the pattern can be obtained in seconds using film plates, compared to 10 to 20 minutes for a pattern to be acquired using the digital scan. Parallel acquisition would, of course, speed up this process immensely.

The glycomes of Caenorhabditis elegans and other model organisms

2002 ◽  
Vol 69 ◽  
pp. 117-134 ◽  
Author(s):  
Stuart M. Haslam ◽  
David Gems ◽  
Howard R. Morris ◽  
Anne Dell
Keyword(s):  
Caenorhabditis Elegans ◽  
Current Knowledge ◽  
Structural Data ◽  
Model Organisms ◽  
Entire Genome ◽  
C Elegans ◽  
The Face ◽  
Area Of Concern ◽  
Nematode Worm

There is no doubt that the immense amount of information that is being generated by the initial sequencing and secondary interrogation of various genomes will change the face of glycobiological research. However, a major area of concern is that detailed structural knowledge of the ultimate products of genes that are identified as being involved in glycoconjugate biosynthesis is still limited. This is illustrated clearly by the nematode worm Caenorhabditis elegans, which was the first multicellular organism to have its entire genome sequenced. To date, only limited structural data on the glycosylated molecules of this organism have been reported. Our laboratory is addressing this problem by performing detailed MS structural characterization of the N-linked glycans of C. elegans; high-mannose structures dominate, with only minor amounts of complex-type structures. Novel, highly fucosylated truncated structures are also present which are difucosylated on the proximal N-acetylglucosamine of the chitobiose core as well as containing unusual Fucα1–2Gal1–2Man as peripheral structures. The implications of these results in terms of the identification of ligands for genomically predicted lectins and potential glycosyltransferases are discussed in this chapter. Current knowledge on the glycomes of other model organisms such as Dictyostelium discoideum, Saccharomyces cerevisiae and Drosophila melanogaster is also discussed briefly.

How do histone modifications contribute to transgenerational epigenetic inheritance in C. elegans?

2020 ◽  
Vol 48 (3) ◽  
pp. 1019-1034 ◽  
Author(s):  
Rachel M. Woodhouse ◽  
Alyson Ashe
Keyword(s):  
Histone Modifications ◽  
Molecular Mechanisms ◽  
Epigenetic Inheritance ◽  
Nematode Caenorhabditis Elegans ◽  
Histone Methyltransferases ◽  
Transgenerational Epigenetic Inheritance ◽  
C Elegans ◽  
Recent Developments ◽  
Gene Regulatory

Gene regulatory information can be inherited between generations in a phenomenon termed transgenerational epigenetic inheritance (TEI). While examples of TEI in many animals accumulate, the nematode Caenorhabditis elegans has proven particularly useful in investigating the underlying molecular mechanisms of this phenomenon. In C. elegans and other animals, the modification of histone proteins has emerged as a potential carrier and effector of transgenerational epigenetic information. In this review, we explore the contribution of histone modifications to TEI in C. elegans. We describe the role of repressive histone marks, histone methyltransferases, and associated chromatin factors in heritable gene silencing, and discuss recent developments and unanswered questions in how these factors integrate with other known TEI mechanisms. We also review the transgenerational effects of the manipulation of histone modifications on germline health and longevity.

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