Production of transgenic live-bearing fish and crustaceans with replication-defective pantropic retroviral vectors

Transgenic fish have been routinely produced by microinjecting or electroporating foreign DNA into one-cell stage embryos or unfertilized eggs. While both techniques are effective in producing transgenic fish species from which unfertilized or newly fertilized eggs can be easily obtained, these techniques are not applicable to live-bearing fish and many crustacean species where unfertilized or newly fertilized eggs are not readily available. In this paper, we describe a new method of introducing foreign DNA into the live-bearing fish, Poeciliposis lucida, and crayfish, Procambarus clarkii, by directly transforming the immature ovary or testis of these animals with replication-defective pantropic retroviral vectors carrying a reporter gene (neo(R)). A significant fraction of the progeny derived from these treated animals contains the neo(R) reporter gene, determined by a PCR-based assay. The PCR-positive individuals were crossed with nontransgenic individuals, and about 50% of the resulting progeny carried the transgene, suggesting that the F(1) animals are germline transgenic. Integration of the transgenes was confirmed by detecting the junction fragments of the genomic DNA associated with transgene constructs. The expression of reporter genes was detected by reverse transcription (RT) PCR assay. These results showed that foreign genes could be reproducibly transferred into live-bearing fish and crustaceans by directly transforming the immature gonads with replication-defective pantropic retroviral vectors.