Abstract:Genome editing in fish has many potential applications in aquaculture and is important for gene functional analyses and genetic engineering breeding. Furthermore, genome editing in aquaculture animals is useful for cultivating new varieties and researching disease resistance and aquaculture growth. However, the difficulty of microinjection and cultivation in mariculture fish embryos seriously hampers the application of genome editing technology. Many methods are used to introduce DNA, RNA, and protein to fish genomes, such as pulsed electric, retroviral transfection, and laser-mediated technologies. Microinjection is the most widely used method. Turbot (Scophthalmus maximus) is one of the most important mariculture fish in Chinese aquaculture. To our knowledge, there have been no reports on the success of the microinjection technique in turbots. In this study, the smyd1 : gfp plasmid (50 ng/µl) with green fluorescent protein (GFP) expression by a 5.3 kb muscle-specific smyd1 promoter fragment was successfully microinjected and expressed in the cytoplasm of newly zygote of turbot at the 1~4 cell stage. The effects of microinjection on the survival rate of turbot embryos were studied. The survival rate of microinjected embryos was lower than that of uninjected embryos. Overall, 120 microinjected fishes (4.8%) survived and 190 (19.0%) uninjected survived. The GFP expression in the embryos and larvae was observed using a fluorescence microscope (Nikon Eclipse 80i). In total, 56% (28/50) of microinjected fishes expressed GFP. A specific GFP-encoding segment (346 bp) was amplified from the DNA of microinjected embryos and larvae by polymerase chain reaction, showing that the GFP gene had been introduced into the turbot embryos and confirming successful microinjection technique of turbot.
