Transgenic Expression and Genome Editing by Electroporation of Zebrafish Embryos
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ORIGINAL ARTICLE
Transgenic Expression and Genome Editing by Electroporation of Zebrafish Embryos Changqing Zhang 1 & Ziheng Ren 1 & Zhiyuan Gong 1 Received: 24 April 2020 / Accepted: 21 July 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract Microinjection is predominantly used to produce genetically modified fish. However, there are certain difficulties involved in some fish species. In this study, we tested an alternative method to produce genetically modified zebrafish by delivering DNA and other materials into embryos by electroporation. We optimized the electroporation conditions of a square wave electroporation system that work efficiently for the introduction of plasmid DNA, recombinant Cas9 nuclease and synthetic dual guide RNAs. Transgenic expression was observed in a wide range of tissues, which is comparable with those obtained by microinjection. We further determined that efficient gene delivery can be achieved during the cleavage stage. This study describes detailed electroporation parameters for gene delivery with high efficiency and low toxicity, providing a novel method to generate transgenic lines and genome editing. Keywords Electroporation . Zebrafish embryos . Transgenesis . CRISPR/Cas9 . Genome editing
Introduction Transgenic fish have been produced mostly through microinjection by delivering genetic materials into fertilized eggs. However, microinjection can be hard to carry out in species with tough chorions, fast development and/or short reproductive seasons (Muller et al. 1993). In such cases, microinjection requires sophisticated manual skills and becomes the limiting step for the production of transgenic lines. Additionally, exogenous genetic materials must be injected at the one- or twocell stage, which could be difficult for some fish species with unclear reproduction patterns, especially in aquaculture. Therefore, alternate gene transfer methods would be essential for genetic engineering. Electroporation is a technique in which short electrical pulses are applied to create microscopic pores on cell membranes through which negatively charged nucleic acids are Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10126-020-09985-0) contains supplementary material, which is available to authorized users. * Zhiyuan Gong [email protected] 1
Department of Biological Sciences, National University of Singapore, 14 Sciences Drive 4, Singapore 117558, Singapore
transferred into the cells. It has been widely used to transform bacteria, yeast and culture cells (Dunham et al. 2018; Kotnik et al. 2015). The use of electroporation to generate transgenic fish has been reported in species such as the channel catfish, common carp, medaka and zebrafish (Powers et al. 1992). Generally, genetic materials can be delivered into fertilized eggs and gametes by different forms of electrical pulses. Sperm-mediated gene transfer by electroporation has been reported in many species, but it is not widely used due to low efficiency. Ferti
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