Gene transfer for Japanese flounder fertilized eggs by particle gun bombardment |
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| Authors: | Ryosuke?Yazawa Ikuo?Hirono Ehchi?Yamamoto Email author" target="_blank">Takashi?AokiEmail author |
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| Institution: | Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Minato, Tokyo 108-8477 and;Tottori Prefectural Fisheries Station, Tomari, Tottori 689-0602, Japan |
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| Abstract: | ABSTRACT: Fish transgenesis has progressed considerably. However, the technique of gene transfer for most marine aquaculture species has not been established. A method to introduce foreign genes into fertilized eggs of Japanese flounder Paralichthys olivaceus by particle gun bombardment was developed by the authors. A recombinant plasmid which contains the Japanese flounder keratin gene promoter linked to the green fluorescence protein (GFP) gene was introduced into fertilized eggs by particle gun bombardments twice at 250 psi. In each experimental group, 25 000–35 000 eggs were treated. However, the survival rate was 12.8% which is lower than that of the control groups (56.8%). Of 2606–5205 the embryos survived for 24 h, 43–61 GFP positive embryos were obtained in one experiment, giving a final gene transfer efficiency of 1.4%. All GFP-positive embryos developed and hatched normally. GFP expression was observed in epithelial tissue throughout the developmental stages. At 3 months after gene transfer, foreign DNA was detected by genome polymerase chain reaction analysis in 37 of 69 fry (53.6%). These results suggest that the particle gun method is an effective method to use with fertilized Japanese flounder eggs. |
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| Keywords: | green fluorescence protein Japanese flounder keratin promoter particle gun transgenic fish |
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