The objective of this research is to develop technologies that will enhance and expand the resources available for performing mutagenesis screens in the mouse. Mutagenesis screens provide the opportunity to identify gene function and generate mouse models to study the genetic basis of human disease and disability. The benefits of the information obtained from The Human and Mouse Genome Projects depend on linking genes with biological function. Toward this end several large scale mutagenesis projects have been initiated in the mouse. It is anticipated that these projects will generate hundreds of new mouse models. However, there is still the need for improved screening methods that will enable mutagenesis applications in the mouse to reach their full potential. We propose a strategy for generating marked chromosomes that permits all classes of mutagenesis screen progeny to be identified throughout gestation and post-natal life. The marker system has the potential for genome-wide application and has been designed to integrate with current mutagenesis screen approaches and recent advances for using germline competent mutagenized ES cells to create mice that harbor either large deletions or single gene mutations. The successful development of this technology will make possible focused mutagenesis screens that can uncover recessive mutations in either two or ultimately one generation. These advances increase opportunities for conducting screens to identify mouse models for the study of development or to recover models that require aging mice, such as cancer. The system takes advantage of recent technologies using the enhanced green fluorescence protein (EGFP) as a ubiquitously expressed transgene, permitting the easy identification of mice and embryos throughout gestation. We plan to design and test a single vector that drives the ubiquitous expression of both EGFP and the red fluorescent (RFP) variant, where each color cassette can be removed independently using site-specific Cre or Flp recombinases. The feasibility of generating a genome-wide resource using this system will be tested through the generation of at least 5 mouse lines derived from stable, randomly integrated red/green ES cells and processing 50 of these ES cell clones to identify their map position in the genome. The availability and facilitated transfer of this technology and these resources to both large centers and individual labs should enhance the range and efficiency of using mutagenesis for functional analysis of the mammalian genome.
