Expression of the antifreeze protein gene in transgenic goldfish (Carassius auratus) and its implication in cold adaptation.

Ocean pout (Macrozoarces americanus) antifreeze protein (AFP) genes (approximately 10(6) copies) were microinjected into the oocytes of goldfish (Carassius auratus). Out of a total of 303 oocytes injected, 235 (77.6%) were normally fertilized after in vitro maturation and insemination, and 136 (57.9%) of the fertilized eggs were hatched. Dot blot analysis of genomic DNA from 2-month-old fish indicated that 30 of the fish (22%) were positive for the AFP transgene. The numbers of integrated AFP genes ranged from a single copy to multiple copies per cell. Two P1 male founders, Y45 and GV16, were crossed separately with a control female, and the inheritance of the AFP transgene was analyzed. Results from Southern blotting and polymerase chain reaction were found to be 7% (5 out of 82) and 58.8% (20 out of 34), respectively, indicating that these two founders are mosaic. The percentage of positive F2 generated from Y45F1 crossed with a control female was 56%, consistent with a Mendelian inheritance. Mature AFP detected by immunoblotting was expressed in both F1 and F2 offspring. These studies indicate that the antifreeze protein gene is successfully transferred and expressed in goldfish. Transgenic goldfish are significantly more cold tolerant than controls when challenged with low temperatures. These studies suggest that the AFP gene may have application in providing cold tolerance in addition to freeze resistance for a variety of fish species.