Transgenic expression in zebrafish embryos with an intact chorion by electroporation and microinjection

•Implemented a novel method for transgenesis of zebrafish embryos combining microinjection and electroporation and developed a high-throughput electroporation system.•Electroporation for multiple zebrafish embryos without removing the chorion is possible by injecting genetic material inside the chorion before application of pulses.•Optimized electrical parameters (Electric pulse amplitude, number of pulses, pulse duration and pulse gap).•Optimized volume and concentration of genetic material injected inside the chorion.•The system can be converted to a high throughput automated transgenesis system for zebrafish embryos. Electroporation is regularly used to deliver agents into cells, including transgenic materials, but it is not used for mutating zebrafish embryos due to the lack of suitable systems, information on appropriate operating parameters, and the challenges posed by the protective chorion. Here, a novel method for gene delivery in zebrafish embryos was developed by combining microinjection into the space between the chorion and the embryo followed by electroporation. This method eliminates the need for chorion removal and injecting into the space between the chorion and embryo eliminates the need for finding and identifying key cell locations before performing an injection, making the process much simpler and more automatable. We also developed a microfluidic electroporation system and optimized electric pulse parameters for transgenesis of embryos. The study provided a novel method for gene delivery in zebrafish embryos that can be potentially implemented in a high throughput transgenesis or mutagenesis system. [Display omitted]