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.

Performing 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 longterm 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.

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