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 electroporating naked DNA into 1-cell-stage embryos or unfertilized eggs. However, these techniques are inapplicable to live-bearing fish and many crustacean species for which unfertilized or newly fertilized eggs are not readily obtainable. In the present study, replication-defective pantropic retroviral vectors carrying a reporter gene (neo(R) or beta -gal) were used to directly transform the immature ovary or testis of a live-bearing fish (Poeciliopsis lucida) and crayfish (Procambarus clarkii). The fraction of the progeny derived from these treated individuals shown to contain the neo(R) reporter gene by all assay based oil polymerase chain reaction (PCR) was significant. 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. Expression of reporter genes was detected by a reverse transcription-nested PCR assay. These results showed that transgenic live-bearing Fish and crustaceans could be easily produced by directly transforming the immature gonads with replication-defective pantropic retroviral vectors.