scholarly journals Electroporation of short hairpin RNAs for rapid and efficient gene knockdown in the starlet sea anemone, Nematostella vectensis

2018 ◽  
Author(s):  
Ahmet Karabulut ◽  
Shuonan He ◽  
Cheng-Yi Chen ◽  
Sean A. McKinney ◽  
Matthew C. Gibson
Keyword(s):  
Large Scale ◽  
Model Organism ◽  
Evolutionary Developmental Biology ◽  
Sea Anemone ◽  
Nematostella Vectensis ◽  
Efficient Gene ◽  
Potential Applications ◽  
Microrna Processing ◽  
Short Hairpin ◽  
Short Hairpin Rnas

ABSTRACTA mechanistic understanding of evolutionary developmental biology requires the development of novel techniques for the manipulation of gene function in phylogenetically diverse organismal systems. Recently, gene-specific knockdown by microinjection of short hairpin RNA (shRNA) has been applied in the sea anemone Nematostella vectensis, a cnidarian model organism. Due to the unusual architecture of the cnidarian microRNA processing pathway, the shRNA approach is unusually effective for sequence-specific knockdown of a gene of interest. However, the time- and labor-intensive process of microinjection limits access to this technique and its application in large scale experiments. To address this issue, here we present an electroporation protocol for shRNA delivery into Nematostella eggs. This method leverages the speed and simplicity of electroporation, enabling users to manipulate gene expression in hundreds of Nematostella eggs or embryos within minutes. We provide a detailed description of the experimental procedure, including reagents, electroporation conditions, preparation of Nematostella vectensis eggs, and follow-up care of experimental animals. Finally, we demonstrate the knockdown of several endogenous and exogenous genes with known phenotypes and discuss the potential applications of this method.

scholarly journals Electroporation of short hairpin RNAs for rapid and efficient gene knockdown in the starlet sea anemone, Nematostella vectensis

2019 ◽  
Vol 448 (1) ◽  
pp. 7-15 ◽  
Author(s):  
Ahmet Karabulut ◽  
Shuonan He ◽  
Cheng-Yi Chen ◽  
Sean A. McKinney ◽  
Matthew C. Gibson
Keyword(s):  
Sea Anemone ◽  
Gene Knockdown ◽  
Nematostella Vectensis ◽  
Efficient Gene ◽  
Short Hairpin ◽  
Short Hairpin Rnas

scholarly journals Large-Scale, High-Throughput Validation of Short Hairpin RNA Sequences for RNA Interference

2006 ◽  
Vol 11 (3) ◽  
pp. 236-246 ◽  
Author(s):  
Laurence H. Lamarcq ◽  
Bradley J. Scherer ◽  
Michael L. Phelan ◽  
Nikolai N. Kalnine ◽  
Yen H. Nguyen ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Strong Support ◽  
Gc Content ◽  
Rapid Identification ◽  
Hairpin Rna ◽  
Rna Sequences ◽  
Short Hairpin ◽  
Short Hairpin Rnas ◽  
Interfering Rna

A method for high-throughput cloning and analysis of short hairpin RNAs (shRNAs) is described. Using this approach, 464 shRNAs against 116 different genes were screened for knockdown efficacy, enabling rapid identification of effective shRNAs against 74 genes. Statistical analysis of the effects of various criteria on the activity of the shRNAs confirmed that some of the rules thought to govern small interfering RNA (siRNA) activity also apply to shRNAs. These include moderate GC content, absence of internal hairpins, and asymmetric thermal stability. However, the authors did not find strong support for positionspecific rules. In addition, analysis of the data suggests that not all genes are equally susceptible to RNAinterference (RNAi).

Starlet Sea Anemone (Nematostella vectensis) 2021 Environmental Summary, Reptile & Aquatics, Stowers Institute for Medical Research v1

2021 ◽  
Author(s):  
Shane C. Miller ◽  
Diana P Baumann ◽  
M. Shane Merryman
Keyword(s):  
North America ◽  
Medical Research ◽  
Environmental Conditions ◽  
Atlantic Coast ◽  
Model Organism ◽  
Environmental Parameters ◽  
Sea Anemone ◽  
Nematostella Vectensis ◽  
Research Results ◽  
Impact Research

The starlet sea anemone (Nematostella vectensis) is an emerging model organism, and we have maintained a colony at the Stowers Institute since 2007. Nematostella are known as a simple sea anemone, related to other cnidarians such as jellyfish and corals. Native to estuarine environments across the Atlantic coast of North America, from Novia Scotia to Florida, they encounter a variety of environmental conditions (e.g., temperature, salinity). Acknowledging that husbandry conditions and environmental parameters can impact research results we provide information about the housing, nutrition, maintenance, and health for our colony of Nematostella. This information will be applicable to any Nematostella housed in the facility in 2021.

scholarly journals Cellular pathways during spawning induction in the starlet sea anemone Nematostella vectensis

2021 ◽  
Author(s):  
Shelly Reuven ◽  
Mieka Rinsky ◽  
Vera Brekhman ◽  
Assaf Malik ◽  
Oren Levy ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Sexual Reproduction ◽  
Fatty Acid Metabolism ◽  
Molecular Mechanisms ◽  
Acid Metabolism ◽  
Light Exposure ◽  
Model Organism ◽  
Sea Anemone ◽  
Nematostella Vectensis ◽  
Minor Effect

AbstractIn cnidarians, long-term ecological success relies on sexual reproduction. The sea anemone Nematostella vectensis, which has emerged as an important model organism for developmental studies, can be induced for spawning by temperature elevation and light exposure. To uncover molecular mechanisms and pathways underlying spawning, we characterized the transcriptome of Nematostella females before and during spawning induction. We identified an array of processes involving numerous receptors, circadian clock components, cytoskeleton, and extracellular transcripts that are upregulated upon spawning induction. Concurrently, processes related to the cell cycle, fatty acid metabolism, and other housekeeping functions are downregulated. Real-time qPCR revealed that light exposure has a minor effect on expression levels of most examined transcripts, implying that temperature change is a stronger inducer for spawning in Nematostella. Our findings reveal the mechanisms that may enable the mesenteries to serve as a gonad-like tissue for the developing oocytes and expand our understanding of sexual reproduction in cnidarians.Summary statementAnalysis of transcriptional changes during spawning induction in Nematostella vectensis, revealed upregulation of processes related to signal perception and cytoskeleton rearrangement and downregulation of fatty acid metabolism and housekeeping processes.

scholarly journals Cellular pathways during spawning induction in the starlet sea anemone Nematostella vectensis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shelly Reuven ◽  
Mieka Rinsky ◽  
Vera Brekhman ◽  
Assaf Malik ◽  
Oren Levy ◽  
...  
Keyword(s):  
Sexual Reproduction ◽  
Molecular Mechanisms ◽  
Light Exposure ◽  
Model Organism ◽  
Sea Anemone ◽  
Nematostella Vectensis ◽  
Minor Effect ◽  
Developmental Studies ◽  
Ecological Success ◽  
A Minor

AbstractIn cnidarians, long-term ecological success relies on sexual reproduction. The sea anemone Nematostella vectensis, which has emerged as an important model organism for developmental studies, can be induced for spawning by temperature elevation and light exposure. To uncover molecular mechanisms and pathways underlying spawning, we characterized the transcriptome of Nematostella females before and during spawning induction. We identified an array of processes involving numerous receptors, circadian clock components, cytoskeleton, and extracellular transcripts that are upregulated upon spawning induction. Concurrently, processes related to the cell cycle, fatty acid metabolism, and other housekeeping functions are downregulated. Real-time qPCR revealed that light exposure has a minor effect on expression levels of most examined transcripts, implying that temperature change is a stronger inducer for spawning in Nematostella. Our findings reveal the potential mechanisms that may enable the mesenteries to serve as a gonad-like tissue for the developing oocytes and expand our understanding of sexual reproduction in cnidarians.

scholarly journals Common Environmental Pollutants Negatively Affect Development and Regeneration in the Sea Anemone Nematostella vectensis Holobiont

2021 ◽  
Vol 9 ◽  
Author(s):  
Sylvia Klein ◽  
Victoria Frazier ◽  
Timothy Readdean ◽  
Emily Lucas ◽  
Erica P. Diaz-Jimenez ◽  
...  
Keyword(s):  
United States ◽  
East Coast ◽  
Environmental Pollutants ◽  
Model Organism ◽  
Potassium Nitrate ◽  
Sea Anemone ◽  
Environmental Stressors ◽  
Nematostella Vectensis ◽  
Early Exposure ◽  
Changing Environments

The anthozoan sea anemone Nematostella vectensis belongs to the phylum of cnidarians which also includes jellyfish and corals. Nematostella are native to United States East Coast marsh lands, where they constantly adapt to changes in salinity, temperature, oxygen concentration and pH. Its natural ability to continually acclimate to changing environments coupled with its genetic tractability render Nematostella a powerful model organism in which to study the effects of common pollutants on the natural development of these animals. Potassium nitrate, commonly used in fertilizers, and Phthalates, a component of plastics are frequent environmental stressors found in coastal and marsh waters. Here we present data showing how early exposure to these pollutants lead to dramatic defects in development of the embryos and eventual mortality possibly due to defects in feeding ability. Additionally, we examined the microbiome of the animals and identified shifts in the microbial community that correlated with the type of water that was used to grow the animals, and with their exposure to pollutants.

scholarly journals Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yang Zhang ◽  
Tuan M. Nguyen ◽  
Xiao-Ou Zhang ◽  
Limei Wang ◽  
Tin Phan ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
High Throughput ◽  
Biological Effect ◽  
Human Hepatocellular Carcinoma ◽  
Guide Rnas ◽  
Rna Targeting ◽  
Short Hairpin ◽  
Short Hairpin Rnas ◽  
Screening Library ◽  
High Throughput Study

AbstractShort hairpin RNAs (shRNAs) are used to deplete circRNAs by targeting back-splicing junction (BSJ) sites. However, frequent discrepancies exist between shRNA-mediated circRNA knockdown and the corresponding biological effect, querying their robustness. By leveraging CRISPR/Cas13d tool and optimizing the strategy for designing single-guide RNAs against circRNA BSJ sites, we markedly enhance specificity of circRNA silencing. This specificity is validated in parallel screenings by shRNA and CRISPR/Cas13d libraries. Using a CRISPR/Cas13d screening library targeting > 2500 human hepatocellular carcinoma-related circRNAs, we subsequently identify a subset of sorafenib-resistant circRNAs. Thus, CRISPR/Cas13d represents an effective approach for high-throughput study of functional circRNAs.

scholarly journals C4 Bacterial Volatiles Improve Plant Health

Pathogens ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 682
Author(s):  
Bruno Henrique Silva Dias ◽  
Sung-Hee Jung ◽  
Juliana Velasco de Castro Oliveira ◽  
Choong-Min Ryu
Keyword(s):  
Plant Growth ◽  
Volatile Compounds ◽  
Large Scale ◽  
Growth Promotion ◽  
Plant Growth Promoting Rhizobacteria ◽  
Plant Health ◽  
Systemic Resistance ◽  
Potential Applications ◽  
Plant Growth Promoting ◽  
Bacterial Volatiles

Plant growth-promoting rhizobacteria (PGPR) associated with plant roots can trigger plant growth promotion and induced systemic resistance. Several bacterial determinants including cell-wall components and secreted compounds have been identified to date. Here, we review a group of low-molecular-weight volatile compounds released by PGPR, which improve plant health, mostly by protecting plants against pathogen attack under greenhouse and field conditions. We particularly focus on C4 bacterial volatile compounds (BVCs), such as 2,3-butanediol and acetoin, which have been shown to activate the plant immune response and to promote plant growth at the molecular level as well as in large-scale field applications. We also disc/ uss the potential applications, metabolic engineering, and large-scale fermentation of C4 BVCs. The C4 bacterial volatiles act as airborne signals and therefore represent a new type of biocontrol agent. Further advances in the encapsulation procedure, together with the development of standards and guidelines, will promote the application of C4 volatiles in the field.

scholarly journals In Vivo Production of RNA Aptamers and Nanoparticles: Problems and Prospects

Molecules ◽  
2021 ◽  
Vol 26 (5) ◽  
pp. 1422
Author(s):  
Ousama Al Shanaa ◽  
Andrey Rumyantsev ◽  
Elena Sambuk ◽  
Marina Padkina
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Large Scale ◽  
Potential Model ◽  
Model Organism ◽  
Rna Aptamers ◽  
Early Stages ◽  
Near Future

RNA aptamers are becoming increasingly attractive due to their superior properties. This review discusses the early stages of aptamer research, the main developments in this area, and the latest technologies being developed. The review also highlights the advantages of RNA aptamers in comparison to antibodies, considering the great potential of RNA aptamers and their applications in the near future. In addition, it is shown how RNA aptamers can form endless 3-D structures, giving rise to various structural and functional possibilities. Special attention is paid to the Mango, Spinach and Broccoli fluorescent RNA aptamers, and the advantages of split RNA aptamers are discussed. The review focuses on the importance of creating a platform for the synthesis of RNA nanoparticles in vivo and examines yeast, namely Saccharomyces cerevisiae, as a potential model organism for the production of RNA nanoparticles on a large scale.

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