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Transgenic Zebrafish by Electroporation

Russell J. Buono and Paul J. Linser, Whitney Marine Laboratory, University of Florida, 9505 Ocean Shore Blvd., St. Augustine, Florida 32086, 904-461-4000


The production of transgenic animals has proven to be a powerful tool in the study of developmentally regulated genes. The traditional method of microinjection of foreign DNA into fertilized embryos is tedious and time consuming. We have used the Bio-Rad Gene Pulser apparatus to generate transgenic fish by electroporation. To establish our technique we used the plasmid construct RSVCAT (Gorman et al., Proc. Natl. Acad. Sci. USA, 79, 6777-6781, 1982). This plasmid uses the Rous sarcoma virus (RSV) promoter to effectively drive expression of chloramphenicol acetyl transferase (CAT) in numerous eukaryotic cell types.


Methods
Fish Maintenance and Egg Collection
Zebrafish (Brachydanio rerio, Figure 1) are obtained from the local pet store and maintained in a 20 gallon aquarium in deionized water (dH20) at 26 C. Fish are fed three times daily: flake food in the morning and at noon, and live hatchling artemia in the afternoon. Fish spawning is induced by the onset of light. Ten to twelve fish are placed in a net breeder with a 1/16th inch nylon mesh net. The net breeder is placed in a 5 gallon aquarium (used only for breeding) with dH20 maintained at 27 C and suspended over a plexiglass container to collect spawned eggs. All tanks are in a room with windows and receive natural sunlight. The fish spawn at first light and eggs are fertilized externally. Fertilized eggs are collected soon after spawning; most embryos are at the two- to four-cell stage. Th
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