Electroporated sperm mediation of a gene transfer system for finfish and shellfish.
We investigated gene transfer in finfish and shellfish via electroporated sperm. The mobility of sperm, the fertilization rate, the hatching rate, gene transfer rate, and abnormality rate of derived embryos were primarily dependent on the voltage level and concentration of DNA during electroporation. Optimal conditions for sperm of each species of aquatic animals can be reached. Genome of the electroporated sperm was analyzed by PCR, and it was shown that an expected-sized product was amplified, corresponding to that of the transgene's amplification. Southern blotting also showed that a positive band located at the same position as the DNA fragment used for the transfer was found in the electroporated sperm after DNase treatment. When the genome isolated from embryos, larvae, juvenile, and adult individuals, all derived from sperm electroporated with foreign DNA molecules, was analyzed by PCR, the existence of foreign DNA was detected in some samples. The integration of the transferred DNA into the genome of transgenic samples was also shown by Southern blot analysis. There was a mosaic distribution of exogenous DNA in a wide variety of tissues analyzed. In addition to CAT activity being positive for the experimental larvae, the transferred GH gene was functional in transgenic finfish and shellfish and resulted in fast-growing transgenic varieties. The overall evidence strongly suggests that the use of electroporated sperm is the simplest yet most efficient approach to perform mass gene transfer in aquacultural animals, including marine mollusks.
Tsai
H J
HJ
Institute of Fisheries Science, National Taiwan University, Taipei.
D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D003531:Cypriniformes / Q000196:embryology / Q000235:genetics*; D004247:DNA / Q000235:genetics; D018274:Electroporation; D018014:Gene Transfer Techniques; D066298:In Vitro Techniques; D008297:Male; D008974:Mollusca / Q000196:embryology / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013094:Spermatozoa / Q000166:cytology*
D004247:DNA