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

Transgenic fish have been routinely produced by microinjecting or electropo rating foreign DNA into one-cell stage embryos or unfertilized eggs. While both techniques are effective in producing transgenic fish species from whi ch unfertilized or newly fertilized eggs can be easily obtained, these tech niques are not applicable to live-bearing fish and many crustacean species where unfertilized or newly fertilized eggs are not readily available. In t his paper, we describe a new method of introducing foreign DNA into the liv e-bearing fish, Poeciliposis lucida, and crayfish, Procambarus clarkii, by directly transforming the immature ovary or testis of these animals with re plication-defective pantropic retroviral vectors carrying a reporter gene ( neo(R)). A significant fraction of the progeny derived from these treated a nimals contains the neo(R) reporter gene, determined by a PCR-based assay. The PCR-positive individuals were crossed with nontransgenic individuals, a nd about 50% of the resulting progeny carried the transgene, suggesting tha t the F-1 animals are germline transgenic. Integration of the transgenes wa s confirmed by detecting the junction fragments of the genomic DNA associat ed 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 crustac eans by directly transforming the immature gonads with replication-defectiv e pantropic retroviral vectors.