Transient expression of RSVCAT in transgenic zebrafish made by electroporation.
We report the fish use of an exponential decay electroporation system to introduce foreign DNA into fertilized zebrafish embryos. The plasmid RSVCAT (Rous sarcoma viral promoter (RSV) upstream from the chloramphenicol acetyltransferase gene (CAT)) was linearized and introduced into fertile zebrafish embryos by electroporation no later than the four-cell stage. Conditions for the procedure were empirically derived, and 68% of the treated animals survived through hatching to at least 6 days after fertilization and well beyond. Dot-blot analysis on DNA extracted from individual hatching fry demonstrated that 65% of the animals tested carried the foreign construct. Enzyme assays on the soluble proteins of treated animals were positive for chloramphenicol acetyltransferase activity. These data demonstrate that the foreign construct was being transiently expressed in the developing tissues of the embryo. The simplicity of this technique will greatly enhance the ability to analyze gene promoter regulation in vivo in transgenic zebrafish. The ability of the electroporated DNA to integrate into the host genome and to generate stable lines of transgenic fish is discussed.
Buono
R J
RJ
Whitney Marine Laboratory, University of Florida, St. Augustine 32086.
Linser
P J
PJ
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics* / Q000378:metabolism; D001358:Avian Sarcoma Viruses / Q000201:enzymology* / Q000235:genetics; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis* / Q000235:genetics; D003584:Cytological Techniques; D015971:Gene Expression Regulation, Enzymologic; D015151:Immunoblotting; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014162:Transfection / Q000379:methods; D015027:Zebrafish / Q000196:embryology / Q000235:genetics* / Q000378:metabolism
D015500:Chloramphenicol O-Acetyltransferase