Two germ cell-driven site-specific recombination systems for the genetic containment of transgenic fish

According to a 2002 study by the National Research Council, the greatest science-based concern facing animal biotechnology is the ecological and environmental impact from the escape or release of transgenic animals. Transgenic fish and insects were of high concern given their ability to escape, disperse and become feral. Current physical and biological methods of containment for domestically raised fish, such as net pens/sea cages and triploidy-induced sterility, are inadequate for the 100% containment required of transgenic fish.Our approach utilized two lines of transgenic zebrafish, a "male" line and a "female" line. In the male line, a germ cell-specific promoter drives flpe recombinase, flanked by loxP sites. In the female line, the gene-of-interest and its promoter are flanked by FRT sites and situated between the germ cell-specific promoter and the cre recombinase coding sequence such that cre expression is blocked. This entire construct is also flanked by loxP sites.We theorized that when these male and female lines were crossed, the progeny would express FLPe in germ cells and trigger excision of the FRT-flanked gene-of-interest. This, in turn, would bring the cre recombinase gene downstream of the germ cell-specific promoter and its expression would cause excision of the loxP-flanked male line construct as well as its own self-excision from the germ cells. Using this approach, it was anticipated that if these fish were outcrossed to wild-type fish, the gametes would not carry any coding transgene and that only two loxP sites would be transmitted to the F2 generation.flpe RNA microinjections into embryos containing the female line construct produced a high proportion of EGFP- progeny, were found to express cre transcript in gonads and produced Cre-mediated excision events in sperm samples. However, when flpe transcript was provided endogenously in the form of a cross with a transgenic male line construct fish, we did not observe excision of the floxed EGFP cassette in F1 progeny as we did in the flpe RNA microinjections. When F1 progeny were outcrossed to wild-type fish, we observed a 1:1 ratio of EGFP+ to EGFP- embryos, suggesting that germline excision by FLPe had not been achieved. A PCR screen on F 2 progeny revealed only two out of 136 fish that were positive for a Cre-mediated excision event. The low efficiency of germline transgene excision could have been affected by several factors including premature self-excision of Cre such that not all floxed sequences were removed, multiple insertions of the female line construct, and poor Cre recombination efficiency related to low temperature.Recently, genetic containment approaches that utilize germ cell-driven site-specific recombinases, such as the Cre/loxP system, to excise transgenes from the germline have been described. However, the paradox of these approaches is that while the transgenic gene-of-interest was removed, the recombinase genes themselves, which are also transgenes, were not excised. We designed and tested a unique genetic containment approach that utilized two germ cell-driven site-specific recombination systems that excised not only the transgenic gene-of-interest, but also the recombinase transgenes themselves, leaving behind two loxP footprints in the germline of transgenic fish.