Gene knockdown via electroporation of short hairpin RNAs in embryos of the marine hydroid Hydractinia symbiolongicarpus

Abstract Analyzing gene function in a broad range of research organisms is crucial for understanding the biological functions of genes and their evolution. Recent studies have shown that short hairpin RNAs (shRNAs) can induce gene-specific knockdowns in two cnidarian species. We have developed a detailed, straightforward, and scalable method to deliver shRNAs into fertilized eggs of the hydrozoan cnidarian Hydractinia symbiolongicarpus via electroporation, yielding effective gene-targeted knockdowns that can last throughout embryogenesis. Our electroporation protocol allows for the transfection of shRNAs into hundreds of fertilized H. symbiolongicarpus eggs simultaneously with minimal embryo death and no long-term harmful consequences on the developing animals. We show RT-qPCR and detailed phenotypic evidence of our method successfully inducing effective knockdowns of an exogenous gene (eGFP) and an endogenous gene (Nanos2), as well as knockdown confirmation by RT-qPCR of two other endogenous genes. We also provide visual confirmation of successful shRNA transfection inside embryos through electroporation. Our detailed protocol for electroporation of shRNAs in H. symbiolongicarpus embryos constitutes an important experimental resource for the hydrozoan community while also serving as a successful model for the development of similar methods for interrogating gene function in other marine invertebrates.

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Gene knockdown via electroporation of short hairpin RNAs in embryos of the marine hydroid Hydractinia symbiolongicarpus

10.1101/2020.04.07.030296 ◽

2020 ◽

Author(s):

Gonzalo Quiroga-Artigas ◽

Alexandrea Duscher ◽

Katelyn Lundquist ◽

Justin Waletich ◽

Christine E. Schnitzler

Keyword(s):

Gene Function ◽

Marine Invertebrates ◽

Larval Settlement ◽

Endogenous Gene ◽

Successful Model ◽

Hydractinia Symbiolongicarpus ◽

Settlement And Metamorphosis ◽

Short Hairpin ◽

Visual Confirmation ◽

Short Hairpin Rnas

AbstractPerforming gene function analyses in a broad range of research organisms is crucial for understanding the biological functions of genes and their evolution. Recent studies have shown that short hairpin RNAs (shRNAs) can induce gene-specific knockdowns in two cnidarian species. We have developed a detailed, straightforward, and scalable method to deliver shRNAs into fertilized eggs of the hydrozoan cnidarian Hydractinia symbiolongicarpus via electroporation, yielding gene-targeted knockdowns that can be assessed throughout embryogenesis, larval settlement, and metamorphosis. Our electroporation protocol allows for the transfection of shRNAs into hundreds of fertilized H.symbiolongicarpus eggs simultaneously with minimal embryo death and no long-term harmful consequences on the developing animals. We show RT-qPCR and detailed phenotypic evidence of our method successfully inducing significant knockdowns of an exogenous gene (eGFP) and an endogenous gene (Nanos2). We also provide visual confirmation of successful shRNA transfection inside embryos through electroporation. This is the first time that electroporation as a delivery system has been developed for Hydractinia. Our detailed protocol for electroporation of shRNAs in H. symbiolongicarpus embryos constitutes an important experimental resource for the hydrozoan community while also serving as a successful model for the development of similar methods for interrogating gene function in other marine invertebrates.

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Publisher Correction: Gene knockdown via electroporation of short hairpin RNAs in embryos of the marine hydroid Hydractinia symbiolongicarpus

Scientific Reports ◽

10.1038/s41598-020-73062-8 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Gonzalo Quiroga-Artigas ◽

Alexandrea Duscher ◽

Katelyn Lundquist ◽

Justin Waletich ◽

Christine E. Schnitzler

Keyword(s):

Gene Knockdown ◽

Hydractinia Symbiolongicarpus ◽

Short Hairpin ◽

Short Hairpin Rnas

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Synthetic Pre-miRNA-Based shRNA as Potent RNAi Triggers

Journal of Nucleic Acids ◽

10.4061/2011/131579 ◽

2011 ◽

Vol 2011 ◽

pp. 1-6 ◽

Cited By ~ 21

Author(s):

Kazuya Terasawa ◽

Kazuharu Shimizu ◽

Gozoh Tsujimoto

Keyword(s):

Biological Activity ◽

Rna Interference ◽

Gene Function ◽

Small Interfering Rnas ◽

Therapeutic Applications ◽

Short Hairpin ◽

Short Hairpin Rnas ◽

Interferon Responses ◽

Rnai Activity ◽

Target Effects

RNA interference (RNAi) is a powerful tool for studying gene function owing to the ease with which it can selectively silence genes of interest, and it has also attracted attention because of its potential for therapeutic applications. Chemically synthesized small interfering RNAs (siRNAs) and DNA vector-based short hairpin RNAs (shRNAs) are now widely used as RNAi triggers. In contrast to expressed shRNAs, the use of synthetic shRNAs is limited. Here we designed shRNAs modeled on a precursor microRNA (pre-miRNA) and evaluated their biological activity. We demonstrated that chemically synthetic pre-miRNA-based shRNAs have more potent RNAi activity than their corresponding siRNAs and found that their antisense strands are more efficiently incorporated into the RNA-induced silencing complex. Although greater off-target effects and interferon responses were induced by shRNAs than by their corresponding siRNAs, these effects could be overcome by simply using a lower concentration or by optimizing and chemically modifying shRNAs similar to synthetic siRNAs. These are challenges for the future.

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Artificial miRNAs are potential gene therapy tools, especially for incurable monogenic disorders

10.31219/osf.io/d5rnm ◽

2021 ◽

Author(s):

Moataz Dowaidar

Keyword(s):

Gene Therapy ◽

Brain Surgery ◽

Native Protein ◽

Intravenous Injections ◽

Monogenic Disorders ◽

Monogenic Diseases ◽

Short Hairpin ◽

The Cost ◽

Short Hairpin Rnas

Well-designed artificial miRNAs (amiRNAs) are as effective as short hairpin RNAs (shRNAs) but produce 10–80 times less siRNA. They enable long-term silencing and are safer than other RNAi triggers. They are suitable instruments for gene therapy techniques, especially for incurable monogenic diseases. In clinical studies, stereotactic injection of AAV5 directly into the striatum is the most effective approach. Intravenous injections would not only make patients more comfortable, but would also reduce the cost of complex brain surgery. In terms of structure, biogenesis, and expression levels, Ami RNAs are more "natural" than other gene therapy methods. They also utilise the cell's native protein machinery and do not produce irreversible alterations, unlike genome editing technologies. The amount of time spent on a technology determines its level of progression. ASOs have an edge in this regard, as seen by the number of authorized medicines. Perhaps RNAi is just around the corner.

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RNA Interference Therapy for Machado–Joseph Disease: Long-Term Safety Profile of Lentiviral Vectors Encoding Short Hairpin RNAs Targeting Mutant Ataxin-3

Human Gene Therapy ◽

10.1089/hum.2018.157 ◽

2019 ◽

Vol 30 (7) ◽

pp. 841-854 ◽

Cited By ~ 5

Author(s):

Clévio Nóbrega ◽

José Miguel Codêsso ◽

Liliana Mendonça ◽

Luís Pereira de Almeida

Keyword(s):

Rna Interference ◽

Safety Profile ◽

Lentiviral Vectors ◽

Short Hairpin ◽

Machado Joseph Disease ◽

Short Hairpin Rnas

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

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057105284342 ◽

2006 ◽

Vol 11 (3) ◽

pp. 236-246 ◽

Cited By ~ 6

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).

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

Genome Biology ◽

10.1186/s13059-021-02263-9 ◽

2021 ◽

Vol 22 (1) ◽

Cited By ~ 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.

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Fossil Record of Parasitism on Marine Invertebrates with Special Emphasis on the Platyceratid-Crinoid Interaction

The Paleontological Society Papers ◽

10.1017/s1089332600001091 ◽

2002 ◽

Vol 8 ◽

pp. 195-210 ◽

Cited By ~ 33

Author(s):

Tomasz K. Baumiller ◽

Forest J. Gahn

Keyword(s):

Fossil Record ◽

Marine Invertebrates ◽

Marine Diversity ◽

Parasitic Interactions ◽

Explicit Criteria ◽

Lower Frequencies

The paleontological literature on marine invertebrates is rich in supposed examples of parasitism and our tabulation shows a nearly even distribution of reported cases through the post-Cambrian Phanerozoic. Slightly lower frequencies characterize the Triassic and Jurassic and higher frequencies the Cretaceous and Tertiary, and the pattern roughly mirrors Sepkoski's (1984) marine diversity curve. The total number of parasitic associations for any geologic period rarely exceeds a dozen, yet few of the reported examples provide explicit criteria distinguishing parasitism from predation, commensalism, or mutualism. We evaluated the published examples using the following criteria: (1) evidence of a long-term relationship between two organisms, (2) benefit of interaction to supposed parasite, and (3) detriment of interaction to the host We found that only in exceptional cases were these criteria fulfilled. One example that provides much information on parasitic interactions involves platyceratids and crinoids and we summarize the evidence for the parasitic interaction between these two groups of organisms.

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