Transgenic live-bearing fish and crustaceans produced by transforming immature gonads with replication-defective pantropic retroviral vectors

Transgenic animals have been routinely produced by microinjecting or electr oporating naked DNA into 1-cell-stage embryos or unfertilized eggs. However , these techniques are inapplicable to live-bearing fish and many crustacea n species for which unfertilized or newly fertilized eggs are not readily o btainable. In tile present study, replication-defective pantropic retrovira l vectors carrying a reporter gene (neo(R) or beta -gal) were used to direc tly transform the immature ovary or testis of a live-bearing fish (Poecilio psis lucida) and crayfish (Procambarus clarkii). The fraction of the progen y derived from these treated individuals shown to contain the neo(R) report er gene by all assay based oil polymerase chain reaction (PCR) was signific ant. The PCR-positive individuals were crossed with nontransgenic individua ls, and about 50% of the resulting progeny carried the transgene, suggestin g that the F-1 animals are germline transgenic. Integration of the transgen es was confirmed by detecting the junction fragments of the genomic DNA ass ociated with transgene constructs. Expression of reporter genes was detecte d by a reverse transcription-nested PCR assay. These results showed that tr ansgenic live-bearing Fish and crustaceans could be easily produced by dire ctly transforming the immature gonads with replication-defective pantropic retroviral vectors.