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.
Highlights
Hydrozoans are members of the phylum Cnidaria, a group that holds a phylogenetic position as sister to all bilaterian animals[1] (Fig. 1A), and can provide insights into the origins of key bilaterian features
Short hairpin RNAs are small, synthetic double-stranded RNA (dsRNA) molecules connected by a hairpin loop that can be used instead of longer dsRNAs to knock down target genes via R NAi17. short hairpin RNAs (shRNAs) are processed to precursor microRNAs through the endogenous RNA interference (RNAi) pathway of transfected c ells18. shRNAs have been widely used to induce gene knockdowns in mammalian cell c ulture[19], in vivo in mammalian m odels[20], and in model systems such as the fruit fly[21] and zebrafish[22]
We reasoned that electroporation trials with Dextran would help to visually demonstrate whether small molecules can be successfully transfected into H. symbiolongicarpus embryos and to test conditions to maximize the survivorship of embryos and overall transfection efficiency
Summary
Hydrozoans are members of the phylum Cnidaria, a group that holds a phylogenetic position as sister to all bilaterian animals[1] (Fig. 1A), and can provide insights into the origins of key bilaterian features. ShRNAs have been widely used to induce gene knockdowns in mammalian cell c ulture[19], in vivo in mammalian m odels[20], and in model systems such as the fruit fly[21] and zebrafish[22] This shRNA-based knockdown approach has recently been used to target a small number of genes in two cnidarian species[23,24,25], capitalizing on the specificity of the cnidarian miRNA p athway[23,26] to achieve robust knockdowns. In a more recent study using H. symbiolongicarpus, shRNAs microinjected in fertilized eggs successfully yielded targeted gene knockdown[25], making shRNAs a promising tool to silence genes in Hydractinia. As an alternative to these delivery methods, electroporation has been widely used by developmental biologists to transfect embryos from different phyla with a range of b iomolecules[27]
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