Transgenic animals are invaluable tools for the elucidation of normal and abnormal biological processes in intact systems. As such they have been used to study a host of health-related processes. Although there are several methods for the production of transgenic animals, most are generated by pronuclear injection or by homologous recombination in embryonic stem (ES) cells. While the pronuclear technology in mice is well developed, it still requires a considerable technical expertise, time, and resources to produce mice by homologous recombination. As an alternative to the production of transgenic animals by traditional blastocyst or pronuclear injections protocols, site-specific recombination systems are being proposed to direct transgenes into predetermined chromosomal sites. One site- specific system which has shown to be functional in mammalian cells in the Cre-loxP composed of a recombinase (Cre) and its DNA recognition site (loxP). We propose to develop a Cre-loxP based system for the site-directed introduction of transgenes into predetermined locations of the mammalian genome. This technology should be applicable to both murine, and non-murine species and would widely benefit the biomedical community. The overall objective of this application include: 1) The identification of mutant loxP sites that favor Cre-mediated integration reaction. A bacterial mutagenesis analysis system will be utilized to determine the effects of different mutations in the loxP sites on the frequency of insertion. 2) Testing the efficiency of the mutant loxP sites favoring the insertion reaction identified in aim 1, as well as mutants previously described by others, will be tested in the WAP locus of mouse embryonic stem cells. Mutant loxP sites will be introduced in the WAP locus by homologous recombination and the frequency of Cre-mediated integration determined. 3) Determining the frequency of Cre-mediated integration by pronuclear injection. As the eventual aim of this application is the development of a site-directed transgenic system that can be used by pronuclear injection we propose to generate a transgenic mouse line containing a loxP-tagged test locus. This transgenic line will be used to determine the factors affecting the frequency of integration of mutant loxP sites when using pronuclear injection. This proposal is the first step in the development of a new, reliable technique for the site-directed modification of the mammalian genome.
