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

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

Scientific Reports ◽

10.1038/s41598-020-69489-8 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Gonzalo Quiroga-Artigas ◽

Alexandrea Duscher ◽

Katelyn Lundquist ◽

Justin Waletich ◽

Christine E. Schnitzler

Keyword(s):

Gene Function ◽

Marine Invertebrates ◽

Endogenous Gene ◽

Successful Model ◽

Hydractinia Symbiolongicarpus ◽

Endogenous Genes ◽

Short Hairpin ◽

Visual Confirmation ◽

Short Hairpin Rnas

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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Bacterial Nucleobases Synergistically Induce Larval Settlement and Metamorphosis in the Invasive MusselMytilopsis sallei

Applied and Environmental Microbiology ◽

10.1128/aem.01039-19 ◽

2019 ◽

Vol 85 (16) ◽

Cited By ~ 1

Author(s):

Jian He ◽

Qi Dai ◽

Yuxuan Qi ◽

Pei Su ◽

Miaoqin Huang ◽

...

Keyword(s):

Marine Invertebrates ◽

Larval Settlement ◽

Aquatic Systems ◽

Substrate Selection ◽

Content Type ◽

Dreissenid Mussels ◽

Settlement And Metamorphosis ◽

Environmental Bacteria ◽

Fouling Organisms ◽

Chemical Mediators

ABSTRACTMarine bacterial biofilms have long been recognized as potential inducers of larval settlement and metamorphosis in marine invertebrates, but few chemical cues from bacteria have been identified. Here, we show that larval settlement and metamorphosis of an invasive fouling mussel,Mytilopsis sallei, could be induced by biofilms of bacteria isolated from its adult shells and other substrates from the natural environment. One of the strains isolated,Vibrio owensiiMS-9, showed strong inducing activity which was attributed to the release of a mixture of nucleobases including uracil, thymine, xanthine, hypoxanthine, and guanine into seawater. In particular, the synergistic effect of hypoxanthine and guanine was sufficient for the inducing activity ofV. owensiiMS-9. The presence of two or three other nucleobases could enhance, to some extent, the activity of the mixture of hypoxanthine and guanine. Furthermore, we determined that bacteria producing higher concentrations of nucleobases were more likely to induce larval settlement and metamorphosis ofM. salleithan were bacteria producing lower concentrations of nucleobases. The present study demonstrates that bacterial nucleobases play an important role in larval settlement and metamorphosis of marine invertebrates. This provides new insights into our understanding of the role of environmental bacteria in the colonization and aggregation of invasive fouling organisms and of the metabolites used as chemical mediators in cross-kingdom communication within aquatic systems.IMPORTANCEInvasive species are an increasingly serious problem globally. In aquatic ecosystems, invasive dreissenid mussels are well-known ecological and economic pests because they appear to effortlessly invade new environments and foul submerged structures with high-density aggregations. To efficiently control exotic mussel recruitment and colonization, the need to investigate the mechanisms of substrate selection for larval settlement and metamorphosis is apparent. Our work is one of very few to experimentally demonstrate that compounds produced by environmental bacteria play an important role in larval settlement and metamorphosis in marine invertebrates. Additionally, this study demonstrates that bacterial nucleobases can be used as chemical mediators in cross-kingdom communication within aquatic systems, which will enhance our understanding of how microbes induce larval settlement and metamorphosis of dreissenid mussels, and it furthermore may allow the development of new methods for application in antifouling.

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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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Bacterial symbionts in animal development: arginine biosynthesis complementation enables larval settlement in a marine sponge

10.1101/2020.08.06.240770 ◽

2020 ◽

Author(s):

Hao Song ◽

Olivia H Hewitt ◽

Sandie M Degnan

Keyword(s):

Nitric Oxide ◽

Marine Sponge ◽

Marine Invertebrates ◽

Sea Water ◽

Larval Settlement ◽

Critical Role ◽

Bacterial Symbionts ◽

Animal Phyla ◽

Settlement And Metamorphosis ◽

Oxide Synthase

SUMMARYLarval settlement and metamorphosis are regulated by nitric oxide (NO) signalling in a wide diversity of marine invertebrates (1-10). It is surprising, then, that in most invertebrates, the substrate for NO synthesis – arginine – cannot be biosynthesized but instead must be exogenously sourced (11). In the sponge Amphimedon queenslandica, vertically-inherited proteobacterial symbionts in the larva are able to biosynthesize arginine (12,13). Here we test the hypothesis that symbionts might provide arginine to the sponge host so that nitric oxide synthase expressed in the larva can produce NO, which induces metamorphosis (8), and the byproduct citrulline (Fig. 1). First, we find support for an arginine-citrulline biosynthetic loop in this sponge larval holobiont using stable isotope tracing. In symbionts, incorporated 13C-citrulline decreases as 13C-arginine increases, consistent with the use of exogenous citrulline for arginine synthesis. In contrast, 13C-citrulline accumulates in larvae as 13C-arginine decreases, demonstrating the uptake of exogenous arginine and its conversion to NO and citrulline. Second, we show that while Amphimedon larvae can derive arginine directly from seawater, normal settlement and metamorphosis can occur in artificial sea water lacking arginine. Together, these results support holobiont complementation of the arginine-citrulline loop and NO biosynthesis in Amphimedon larvae, suggesting a critical role for bacterial symbionts in the development of this marine sponge. Given that NO regulates settlement and metamorphosis in diverse animal phyla (1-10) and arginine is procured externally in most animals (11), we propose that symbionts may play a equally critical regulatory role in this essential life cycle transition in other metazoans.

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I Feel That! Fluid Dynamics and Sensory Aspects of Larval Settlement across Scales

10.1093/oso/9780198786962.003.0013 ◽

2018 ◽

Cited By ~ 3

Keyword(s):

Fluid Dynamics ◽

Marine Invertebrates ◽

Larval Settlement ◽

External Environment ◽

Life Cycles ◽

Decision Making Process ◽

Larval Biology ◽

Sea Floor ◽

External Cues ◽

Juvenile Habitat

A commonality among oceanic life cycles is a process known as settlement, where dispersing propagules transition to the sea floor. For many marine invertebrates, this transition is irreversible, and therefore involves a crucial decision-making process through which larvae evaluate their juvenile habitat-to-be. In this chapter, we consider aspects of the external environment that could influence successful settlement. Specifically, we discuss water flow across scales, and how larvae can engage behaviors to influence where ocean currents take them, and enhance the likelihood of their being carried toward suitable settlement locations. Next, we consider what senses larvae utilize to evaluate their external environment and properly time such behavioral modifications, and settlement generally. We hypothesize that larvae integrate these various external cues in a hierarchical fashion, with differing arrangements being employed across ontogeny and among species. We conclude with a brief discussion of the future promises of larval biology, ecology, and evolution.

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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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