GENERATION OF RADIATION-INDUCED DELETION COMPLEXES IN THE MOUSE GENOME USING EMBRYONIC STEM-CELLS

As the genetic and physical mapping stage of the Human Genome Project nears completion, the focus is shifting toward the development of tech nologies for high-throughput analysis of gene function. Whereas DNA se quencing will enable the assignment of presumed function to a large nu mber of genes in mice and humans, it is clear that the great majority of genes will have to be evaluated in vivo to accurately assess their role in a complex organism. While gene targeting in mouse embryonic st em (ES) cells is the current method of choice for the characterization of gene function in mice, it remains relatively labor intensive and l acks the throughput required for analysis of genome function on a larg e scale. Alternative methods of efficient mutagenesis will clearly be required for this task. Chromosomal deletions are powerful tools in th e genetic analysis of complex genomes, enabling the systematic identif ication and localization of functional units along defined chromosomal regions. Not only are deletions useful for the identification of gene tic functions, but they serve as mapping reagents for existing mutatio ns or traits. While their use has been an essential tool in Drosophila genetics, classical mutagenesis in mice has been logistically impract ical for generating deletions. We have previously described an efficie nt method for generating radiation-induced deletion complexes at defin ed regions in the genome using ES cells. In this article, we detail th e methodological aspects of this technology and describe the applicati ons of chromosomal deletions for characterizing gene function in ways that make optimal use of the information generated by the first stage of the Genome Project. (C) 1997 Academic Press.