scholarly journals Single-copy Knock-In Loci for Defined Gene Expression in C. elegans

2018 ◽  
Author(s):  
Carlos G Silva-Garcia ◽  
Caroline Heintz ◽  
Sneha Dutta ◽  
Nicole M Clark ◽  
Anne Lanjuin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Caenorhabditis Elegans ◽  
Cdna Sequence ◽  
Single Copy ◽  
Tissue Specific ◽  
C Elegans

We have generated a single-copy knock-in loci for defined gene expression (SKI LODGE) system to insert any cDNA by CRISPR/Cas9 at defined safe harbors in the Caenorhabditis elegans genome. Utilizing a single crRNA guide, which also acts as a Co-CRISPR enrichment marker, any cDNA sequence can be introduced as a single integrated copy, regulated by different tissue-specific promoters. The SKI LODGE system provides a fast, economical and effective approach for generating single-copy non-silenced transgenes in C. elegans.

scholarly journals The comprehensive transcriptional analysis in Caenorhabditis elegans by integrating ChIP-seq and gene expression data

2014 ◽  
Vol 96 ◽  
Author(s):  
KAN HE ◽  
JIAOFANG SHAO ◽  
ZHONGYING ZHAO ◽  
DAHAI LIU
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Caenorhabditis Elegans ◽  
Gene Expression Data ◽  
Target Genes ◽  
Model Systems ◽  
Transcriptional Analysis ◽  
Expression Data ◽  
Tissue Specific ◽  
C Elegans

SummaryThe fundamental step of learning transcriptional regulation mechanism is to identify the target genes regulated by transcription factors (TFs). Despite numerous target genes identified by chromatin immunopre-cipitation followed by high-throughput sequencing technology (ChIP-seq) assays, it is not possible to infer function from binding alone in vivo. This is equally true in one of the best model systems, the nematode Caenorhabditis elegans (C. elegans), where regulation often occurs through diverse TF binding features of transcriptional networks identified in modENCODE. Here, we integrated ten ChIP-seq datasets with genome-wide expression data derived from tiling arrays, involved in six TFs (HLH-1, ELT-3, PQM-1, SKN-1, CEH-14 and LIN-11) with tissue-specific and four TFs (CEH-30, LIN-13, LIN-15B and MEP-1) with broad expression patterns. In common, TF bindings within 3 kb upstream of or within its target gene for these ten studies showed significantly elevated level of expression as opposed to that of non-target controls, indicated that these sites may be more likely to be functional through up-regulating its target genes. Intriguingly, expression of the target genes out of 5 kb upstream of their transcription start site also showed high levels, which was consistent with the results of following network component analysis. Our study has identified similar transcriptional regulation mechanisms of tissue-specific or broad expression TFs in C. elegans using ChIP-seq and gene expression data. It may also provide a novel insight into the mechanism of transcriptional regulation not only for simple organisms but also for more complex species.

scholarly journals An efficient FLP-based toolkit for spatiotemporal control of gene expression in Caenorhabditis elegans

2017 ◽  
Author(s):  
Celia María Muñoz-Jiménez ◽  
Cristina Ayuso ◽  
Agnieszka Dobrzynska ◽  
Antonio Torres ◽  
Patricia de la Cruz Ruiz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Caenorhabditis Elegans ◽  
Developmental Stages ◽  
Model Organism ◽  
Cell Types ◽  
Single Copy ◽  
Conditional Knockout ◽  
Control Of Gene Expression ◽  
C Elegans ◽  
Cell Ablation

AbstractSite-specific recombinases are potent tools to regulate gene expression. In particular, the Cre and FLP enzymes are widely used to either activate or inactivate genes in a precise spatiotemporal manner. Both recombinases work efficiently in the popular model organism Caenorhabditis elegans but their use in this nematode is still only sporadic. To increase the utility of the FLP system in C. elegans we have generated a series of single-copy transgenic strains that stably express an optimized version of FLP in specific tissues or by heat induction. We show that recombination efficiencies reach 100 percent in several cell types, such as muscles, intestine and serotonin producing neurons. Moreover, we demonstrate that most promoters drive recombination exclusively in the expected tissues. As examples of the potentials of the FLP lines we describe novel tools for induced cell ablation by expression of the PEEL-1 toxin and a versatile FLP-out cassette for generation of GFP-tagged conditional knockout alleles. Together with other recombinase-based reagents created by the C. elegans community this toolkit increases the possibilities for detailed analyses of specific biological processes at developmental stages inside intact animals.

Transfer and tissue-specific accumulation of components in embryos of Caenorhabditis elegans and dolichura: in vivo analysis with a low-cost signal

Development ◽  
1992 ◽  
Vol 114 (2) ◽  
pp. 317-330 ◽  
Author(s):  
O. Bossinger ◽  
E. Schierenberg
Keyword(s):  
Caenorhabditis Elegans ◽  
Low Cost ◽  
Free Living ◽  
Tissue Specific ◽  
C Elegans ◽  
Specific Accumulation ◽  
In Vivo Analysis

The pattern of autofluorescence in the two free-living namatodes Rhabditis dolichura and Caenorhabditis compared. In C. elegans, during later embryogenesis cells develop a typical bluish autofluorescence as illumination, while in Rh. dolichura a strong already present in the unfertilized egg. Using a new,

scholarly journals Temporal Transcriptomics of Gut Escherichia coli in Caenorhabditis elegans Models of Aging

2021 ◽  
Author(s):  
Joshua D. Brycki ◽  
Jeremy R. Chen See ◽  
Gillian R. Letson ◽  
Cade S. Emlet ◽  
Lavinia V. Unverdorben ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Caenorhabditis Elegans ◽  
Life Span ◽  
Wild Type ◽  
Health Span ◽  
Content Type ◽  
C Elegans ◽  
Evolutionarily Conserved

Previous research has reported effects of the microbiome on health span and life span of Caenorhabditis elegans , including interactions with evolutionarily conserved pathways in humans. We build on this literature by reporting the gene expression of Escherichia coli OP50 in wild-type (N2) and three long-lived mutants of C. elegans .

scholarly journals Tissue-Specific Transcription Footprinting Using RNA PoI DamID (RAPID) in Caenorhabditis elegans

Genetics ◽  
2020 ◽  
Vol 216 (4) ◽  
pp. 931-945 ◽  
Author(s):  
Georgina Gómez-Saldivar ◽  
Jaime Osuna-Luque ◽  
Jennifer I. Semple ◽  
Dominique A. Glauser ◽  
Sophie Jarriault ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Rna Polymerase ◽  
Cell Types ◽  
Neuroendocrine Cells ◽  
Cell Physiology ◽  
Specific Gene ◽  
Cell Content ◽  
Tissue Specific ◽  
C Elegans ◽  
Active Transcription

Differential gene expression across cell types underlies development and cell physiology in multicellular organisms. Caenorhabditis elegans is a powerful, extensively used model to address these biological questions. A remaining bottleneck relates to the difficulty to obtain comprehensive tissue-specific gene transcription data, since available methods are still challenging to execute and/or require large worm populations. Here, we introduce the RNA Polymerase DamID (RAPID) approach, in which the Dam methyltransferase is fused to a ubiquitous RNA polymerase subunit to create transcriptional footprints via methyl marks on the DNA of transcribed genes. To validate the method, we determined the polymerase footprints in whole animals, in sorted embryonic blastomeres and in different tissues from intact young adults by driving tissue-specific Dam fusion expression. We obtained meaningful transcriptional footprints in line with RNA-sequencing (RNA-seq) studies in whole animals or specific tissues. To challenge the sensitivity of RAPID and demonstrate its utility to determine novel tissue-specific transcriptional profiles, we determined the transcriptional footprints of the pair of XXX neuroendocrine cells, representing 0.2% of the somatic cell content of the animals. We identified 3901 candidate genes with putatively active transcription in XXX cells, including the few previously known markers for these cells. Using transcriptional reporters for a subset of new hits, we confirmed that the majority of them were expressed in XXX cells and identified novel XXX-specific markers. Taken together, our work establishes RAPID as a valid method for the determination of RNA polymerase footprints in specific tissues of C. elegans without the need for cell sorting or RNA tagging.

scholarly journals Inducible Systemic RNA Silencing in Caenorhabditis elegans

2003 ◽  
Vol 14 (7) ◽  
pp. 2972-2983 ◽  
Author(s):  
Lisa Timmons ◽  
Hiroaki Tabara ◽  
Craig C. Mello ◽  
Andrew Z. Fire
Keyword(s):  
Rna Interference ◽  
Caenorhabditis Elegans ◽  
Rna Silencing ◽  
Molecular Mechanisms ◽  
Normal Animal ◽  
Cell Types ◽  
Animal Cells ◽  
Tissue Specific ◽  
C Elegans ◽  
Systemic Rna

Introduction of double-stranded RNA (dsRNA) can elicit a gene-specific RNA interference response in a variety of organisms and cell types. In many cases, this response has a systemic character in that silencing of gene expression is observed in cells distal from the site of dsRNA delivery. The molecular mechanisms underlying the mobile nature of RNA silencing are unknown. For example, although cellular entry of dsRNA is possible, cellular exit of dsRNA from normal animal cells has not been directly observed. We provide evidence that transgenic strains of Caenorhabditis elegans transcribing dsRNA from a tissue-specific promoter do not exhibit comprehensive systemic RNA interference phenotypes. In these same animals, modifications of environmental conditions can result in more robust systemic RNA silencing. Additionally, we find that genetic mutations can influence the systemic character of RNA silencing in C. elegans and can separate mechanisms underlying systemic RNA silencing into tissue-specific components. These data suggest that trafficking of RNA silencing signals in C. elegans is regulated by specific physiological and genetic factors.

scholarly journals KH domain containing RNA-binding proteins coordinate with microRNAs to regulate Caenorhabditis elegans development

2021 ◽  
Vol 11 (2) ◽  
Author(s):  
Dustin Haskell ◽  
Anna Zinovyeva
Keyword(s):  
Gene Expression ◽  
Caenorhabditis Elegans ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Regulate Gene Expression ◽  
C Elegans ◽  
Binding Domains ◽  
Kh Domain ◽  
Regulate Gene

Abstract MicroRNAs (miRNAs) and RNA-binding proteins (RBPs) regulate gene expression at the post-transcriptional level, but the extent to which these key regulators of gene expression coordinate their activities and the precise mechanisms of this coordination are not well understood. RBPs often have recognizable RNA binding domains that correlate with specific protein function. Recently, several RBPs containing K homology (KH) RNA binding domains were shown to work with miRNAs to regulate gene expression, raising the possibility that KH domains may be important for coordinating with miRNA pathways in gene expression regulation. To ascertain whether additional KH domain proteins functionally interact with miRNAs during Caenorhabditis elegans development, we knocked down twenty-four genes encoding KH-domain proteins in several miRNA sensitized genetic backgrounds. Here, we report that a majority of the KH domain-containing genes genetically interact with multiple miRNAs and Argonaute alg-1. Interestingly, two KH domain genes, predicted splicing factors sfa-1 and asd-2, genetically interacted with all of the miRNA mutants tested, whereas other KH domain genes showed genetic interactions only with specific miRNAs. Our domain architecture and phylogenetic relationship analyses of the C. elegans KH domain-containing proteins revealed potential groups that may share both structure and function. Collectively, we show that many C. elegans KH domain RBPs functionally interact with miRNAs, suggesting direct or indirect coordination between these two classes of post-transcriptional gene expression regulators.

scholarly journals Rapid self-selecting and clone-free integration of transgenes into engineered CRISPR safe harbor locations in Caenorhabditis elegans

2020 ◽  
Author(s):  
Zachary C. Stevenson ◽  
Megan J. Moerdyk-Schauwecker ◽  
Brennen Jamison ◽  
Patrick C. Phillips
Keyword(s):  
Caenorhabditis Elegans ◽  
Single Copy ◽  
False Positives ◽  
Model Organisms ◽  
Screening Methods ◽  
Safe Harbor ◽  
Guide Rna ◽  
C Elegans ◽  
Pcr Products ◽  
Transgenic Lines

AbstractPrecision genome editing for model organisms has revolutionized functional analysis and validation of a wide variety of molecular systems. To date, the capacity to insert transgenes into the model nematode Caenorhabditis elegans has focused on utilizing either transposable elements or CRISPR-based safe harbor strategies. These methods require laborious screening processes that often result in false positives from heritable extrachromosomal arrays or rely on co-CRISPR markers to identify likely edited individuals. As a result, verification of transgene insertion requires anti-array selection screening methods or extensive PCR genotyping respectively. These approaches also rely on cloning plasmids for the addition of transgenes. Here, we present a novel safe harbor CRISPR-based integration strategy that utilizes engineered insertion locations containing a synthetic guide RNA target and a split-selection system to eliminate false positives from array formation, thereby providing integration-specific selection. This approach allows the experimenter to confirm an integration event has taken place without molecular validation or anti-array screening methods, and is capable of producing integrated transgenic lines in as little as five days post-injection. To further increase the speed of generating transgenic lines, we also utilized the C. elegans native homology-based formation of extra-chromosomal arrays to assemble transgenes in-situ, removing the cloning step. We show that complete transgenes can be made and inserted into our split-selection safe harbor locations starting from PCR products, providing a clone-free and molecular-validation-free strategy for single-copy transgene integration. Overall, this combination of approaches provides an economical and rapid system for generating highly reproducible complex transgenics in C. elegans.

scholarly journals New strains for tissue-specific RNAi studies in Caenorhabditis elegans

2018 ◽  
Author(s):  
Jason S. Watts ◽  
Henry F. Harrison ◽  
Shizue Omi ◽  
Quentin Guenthers ◽  
James Dalelio ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Caenorhabditis Elegans ◽  
Gene Function ◽  
Target Genes ◽  
Resistant Mutant ◽  
Knockout Mutant ◽  
Tissue Specific ◽  
Double Stranded Rna ◽  
C Elegans ◽  
Insight Into

AbstractRNA 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. 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. By measuring changes in the fatty acid products of the desaturase enzymes that synthesize PUFAs, 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 thus is more appropriate for construction of a robust tissue-specific RNAi strains. Using newly constructed strains in the rde-1(null) background, we found considerable desaturase activity in intestinal, epidermal, and germline tissues, but not in muscle. The RNAi-specific strains reported in this study will be useful tools for C. elegans researchers studying a variety of biological processes.

scholarly journals Fabrication of sharp silicon arrays to wound Caenorhabditis elegans

2019 ◽  
Author(s):  
Jérôme Belougne ◽  
Igor Ozerov ◽  
Céline Caillard ◽  
Frédéric Bedu ◽  
Jonathan J. Ewbank
Keyword(s):  
Gene Expression ◽  
Caenorhabditis Elegans ◽  
Optical Lithography ◽  
Wet Etching ◽  
C Elegans ◽  
Genes Encoding ◽  
Nematode Cuticle ◽  
Peptide Gene ◽  
Silicon Arrays ◽  
Regular Arrays

ABSTRACTUnderstanding how animals respond to injury and how wounds heal remains a challenge. These questions can be addressed using genetically tractable animals, including the nematode Caenorhabditis elegans. Given its small size, the current methods for inflicting wounds in a controlled manner are demanding. To facilitate and accelerate the procedure, we fabricated regular arrays of pyramidal features (“pins”) sharp enough to pierce the tough nematode cuticle. The pyramids were made from monocrystalline silicon wafers that were micro-structured using optical lithography and alkaline wet etching. The fabrication protocol and the geometry of the pins, determined by electron microscopy, are described in detail. Upon wounding, C. elegans expresses genes encoding antimicrobial peptides. A comparison of the induction of antimicrobial peptide gene expression using traditional needles and the pin arrays demonstrates the utility of this new method.

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