DESCRIPTION: (Applicant's abstract) This proposal aims to develop efficient methods to generate series of targeted large deletions in the genome of murine embryonic stem cells thus allowing the posterior generation of transgenic mouse lines carrying such deletions. These mutant lines will facilitate the functional characterization of the mammalian genome in a way that will complement the primary sequence and other molecular information that is becoming increasingly available as a result of the Genome Projects. This project will attempt to improve on existing chromosomal engineering (CE) technology to lower the cost in terms of money, effort, and time necessary to generate meaningful targeted deletions. Specifically, cloned DNA in a genomic region of interest will be used to generate the targeting vectors necessary to generate a primary targeted deletion (PTD) using an improved method that obviates half the investment used in conventional CE. The next improvement will be the development of a very efficient method to scan the PTD through the generation of "sub-deletions." The size of the sub-deletions will be defined as a midpoint between not to big that the phenotype is too complex too study, and not to small that the number of subdeletions required to scan a given PTD is too large and thus impractical. The general idea being that the sub-deletion scanning will eventually be used extensively if a phenotype justifies the further exploration of the PTD. The proposed methods will applied to five genomic regions with the only intent of testing the feasibility and robustness of the protocols. If successful, the benefits for further characterization of the mouse genome will be significant. A reasonable effort could probably be justified to carry out functional scanning of the mammalian genome since the cost could be brought down significantly and the importance of any given genomic region (and thus the justification to invest resources) could be defined early in the research.