On the strengths of forward genetics and embryology the zebrafish Danio rerio has become an ideal system for the study of early vertebrate development. However, additional tools will be needed to perform more sophisticated analyses and to successfully carry this model into new areas of study such as adult physiology, cancer, and aging. In this regard, one area of technology that is in need of expansion is that of transgenesis and targeted modification of the zebrafish genome. Genome engineering strategies employing site-specific recombinase (SSR) systems such as Cre/lox and Flp/FRT have become invaluable to the study of gene function in the mouse and are now being exploited in Drosophila as well. The goal of this project is to assess the utility of another such SSR, the integrase encoded by the Streptomyces bacteriophage phiC31, for manipulation of the zebrafish genome. The phiC31 integrase promotes recombination between an attachment site in the phage (attP) and another on the bacterial chromosome (attB). One difference between phiC31 and the other systems is that the phiC31 integrase functions unidirectionally, i.e. the products of the recombination between attB and attP are not themselves substrates for the integrase, making this system potentially more efficient for integration of transgenes and for certain intramolecular reactions. In this study, the feasibility of both intermolecular and intramolecular recombination strategies in zebrafish embryos will be explored.
The specific aims will be 1) to determine if phiC31 integrase can mediate precise and efficient insertion of attB-bearing vectors into the zebrafish genome via endogenous (pseudo-attP) sequences, 2) to determine if phiC31 integrase can mediate precise and efficient integration into a zebrafish line transgenic for an attP site, and 3) to determine if phiC31 integrase can be used to excise a transgene cassette flanked by an attB and an attP site. These studies will establish experimental conditions and tangible resources such as transgenic zebrafish lines that will make this a useful approach for the research community.
This research aims to establish new technology for generation and modification of transgenic zebrafish. These tools will advance the usefulness of this model organism as a model for human development and disease. ? ? ?
| Lister, James A (2011) Use of phage ?C31 integrase as a tool for zebrafish genome manipulation. Methods Cell Biol 104:195-208 |
| Lister, James A (2010) Transgene excision in zebrafish using the phiC31 integrase. Genesis 48:137-43 |
