When a fragment of genomic DNA is introduced into a mammalian cell, it can locate and recombine with endogenous homologous sequences. This type of homologous recombination is known as gene targeting by operating embryonic stem cells. As an alternative to embryonic stem cells, cultured somatic cells offer the possibility of producing cloned aquatic animals with targeted genetic manipulations by nuclear transfer. Attempts to produce cloned animals for commercial purposes have been made in cattle, sheep, rabbit, pig, and zebrafish, but were not successful in commercial aquatic animals. Our laboratory is focusing on platform technology development and applications to aquaculture.

1. Development of Site-specific Recombination in Fish Cells and Zebrafish
Recent advances using the Cre-loxP and Flp-FRT systems have now made it possible to generate “clean” germline mutations following a single gene targeting event, as well as to activate and inactivate genes in a condi- tional manner in living mice. These techniques have not yet been applied to fish systems. The techniques not only can target gene mutations which are induced in spatially and temporally restricted fashions, but lineage tracers can also be activated in specific progenitor populations to chart cell fates directly in wildtype and mutant animals. We have chosen two targeting genes: zebrafish IGFBP-2 (chromosome 22) and IGFBP-3. These two genes span around 20 kb in the zebrafish genome. We then studied the features of IGFBP-2 and IGFBP-3 promoter functions, and we now know the expression types in embryos driven by the GFP gene. The next step will be targeting these two genes in zebrafish cells, then carrying out nuclear transfer to study the knockout gene function in zebrafish system.

2. Application of Antimicrobial Peptide to Aquatic Diseases Resistance in Shrimp and Fish
In recent years, hundreds of naturally occurring peptide antibiotics have been discovered based on their ability to inhibit the growth of microbial pathogens. These antimicrobial peptides (AMPs) participate in the innate immune response by providing a rapid first-line defense against infection. To address the need for new therapies to combat resistant organisms, we are refocusing the discovery efforts on developing novel agents with new mechanisms of action by a knock-in technique. The hope is that with improvements in rapidly emerging technologies including gene transfer technology and transgenic aquatic animals, pathogen challenge will occur. These technologies should aid in the identification of novel AMPs as drug targets and compounds with unique mechanisms of action other than those currently provided by traditional antibiotics. We are investigating the roles of AMPs and interferon in aquatic animals using a transgenic approach.

3. Developing Biosafety Technology of Genetically Modified Aquatic Organisms by Site-specific Recombination
We cannot seek permission to market transgenic fish; the main reason is the lack of a field test research area and because no basic research technology has been developed to evaluate their biosafety. Sterility is a necessary adjunct to the exploitation of transgenic fish unless completely secure land-locked facilities are available. In addition, sterility is an important parameter in its own right, even aside from its use for containment of genetically modified fish. The problems posed by the escape of transgenic fish from sea cages and their interbreeding with wild populations in adjacent rivers are well known, and problems with different farmed fish species could at least be partially reduced by the use of sterile fish. Sterility in fish can be achieved by two different routes, namely ploidy manipulation to produce sterile triploids, or the use of transgenesis to achieve gene “knockout” or gene “knockdown” by an RNAi approach with the zebrafish GtH gene.