Optimization of in vivo electroporation and comparison to microinjection as delivery methods for transgenesis in zebrafish (Danio rerio). Generation of a new neuronal zebrafish line

• Autor(a) principal: Almeida, Inês Ferreira de
• Data de Publicação: 2022
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/132850
• topic: Zebrafish embryos; Transgenesis; Microinjection; Electroporation; Genetically encoded fluorescent reporters; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

Transgenic zebrafish are important models for biomedical research. There are several technologies available for the generation of transgenics and for genome editing. However, methods for the delivery of exogenous components remain limited. In Zebrafish, the most used method is microinjection, which requires sophisticated technical skills and presents a low integration rate of large constructs. Alternatively, a few studies reported the use of electroporation as a delivery method for the generation of transgenic zebrafish; however, these protocols contain some limitations that reduce their widespread applicability. To overcome this, we based on the most recent published work reporting electroporation in zebrafish embryos, to implement optimizations in order to increase the number of embryos electroporated, the efficiency of plasmid DNA delivery and its integration in the germline. Electroporation rounds of 30 one-cell stage zebrafish embryos with 300 ng/uL of plasmid DNA in PBS using 35 V poring pulse and 5 V transfer pulse yielded the highest survival and efficiency. Compared to microinjection, the optimized electroporation protocol achieved similar fluorescence intensity and expression pattern, opening the way to becoming a practical and efficient alternative to microinjection. In parallel, a new calcium indicator pan-neuronal transgenic zebrafish line, elalv3:GCaMP6fEF05 was generated, through microinjection into one-cell stage zebrafish embryos, followed by 3 rounds of fish crosses, screens, selection and raising. The improvement of delivery methods, such as electroporation, will expand the generation of new zebrafish lines for the study of developmental and molecular biology that ultimately allows the exploration of new human therapeutic avenues.