Direct DNA delivery into zebrafish embryos employing tissue culture techniques.
The production of transfected fish embryos requires expertise in injecting the fertilized eggs and/or expensive equipment for electroporation or microprojectiles. This article demonstrates that by exposure to DNA constructs conjugated with transfecting reagents dechorionated Danio rerio embryos are capable of acquiring extracellular DNA and expressing reporter genes. Embryos incubated with pCMVluc complexed with GeneJammer or GenePORTER expressed luciferase 24-48 h after exposure. pCMVGFP DNA mixed with the same agents generated embryos that exhibited differential patterns of expression of green fluorescent protein (GFP). Embryonic development varied depending on the procedure employed and the reporter gene utilized. Expression of the luciferase gene did not interfere with the subsequent development of the embryos. In contrast, the embryos expressing a high level of GFP were affected, probably due to a very active promoter. These results demonstrate the ease of obtaining transfected fish embryos, which facilitate the mass production of new genotypes and extend the procedure to laboratories with limited resources.
Sussman
R
R
Marine Biological Laboratory, Marine Resources Center, Woods Hole, Massachusetts 02543, USA. rsussman@marinebio.mbl.edu
D000818:Animals; D046508:Culture Techniques / Q000379:methods; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000378:metabolism*; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics
D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; D008156:Luciferases