Vertebrate gene targeting has so far been possible only in the mouse. Comparative studies of similar mutations in other vertebrates would allow a more complete analysis useful for establishing the importance of conserved genes implicated in fundamental processes of human development and disease. Because the zebrafish embryo is nearly transparent and easily observed during development, it is an excellent model of vertebrate embryogenesis. However, efficient transgenesis of the zebrafish has not yet been established primarily due to the lack of vector systems capable of high level integrated reporter gene expression screenable throughout the life cycle. A non viral vector, pFRMwg, has been designed which stably expresses the green fluorescent protein (GFP) almost ubiquitously throughout the zebrafish. In addition, transgenic techniques have been optimized to allow screening of homologous recombination events in the living embryo. In the proposed study, this vector system will be modified so that an integrated tester locus containing a blue mutation of GFP can be targeted by microinjection of the 1-cell embryo with green GFP vectors and screened for green GFP expression during development. The frequency of this in vivo gene targeting will be tested and optimized as the variables of vector design and the type of embryos chosen to be microinjected are manipulated. The goal will be to establish the conditions for efficient germ line transfer of gene targeting events. Human gene therapy offers a tremendous potential for both the cure and prevention of disease. Again, the primary animal model has been the mouse, and several limitations have become apparent in this system: expression of gene therapy vectors can not be easily screened in live mice, and the potential of unintended germ line transfer can only be tested on a large scale by PCR of male gametes. Additionally, long term persistence of expression (e.g. 1 year) is yet to be shown. Using the pFRMwg vector as a marker for gene therapy in zebrafish, expression can be scored in living fish. In this study, persistence and localization of this expression will be tested as a function of the variables of DNA dose, DNA assimilation cofactors and route of administration and finally correlated with the state of the vector DNA over time. The goals are to achieve persistent high level expression and eventually to estimate the potential of germ line transfer after a particular treatment.
