The immediate goal of this proposal is to develop a method for creating deletion complexes at defined locations in the genome of mice, using embryonic stem (ES) cell technology. The mouse t complex region of chromosome 17 will be used as the test region. The t complex region contains genes affecting spermatogenesis and early development. It also includes the major histocompatibility complex. Two loci along the t complex have been chosen to make DNA targeting constructs containing both positive and negative selectable markers. Targeted Fl hybrid ES cells will be subjected to gamma irradiation under conditions we have shown-to maintain their germ cell-colonizing potential. It is known that gamma irradiation induces interstitial chromosome deletions. Deletions that cover the integration site of the targeting construct will remove the negative selectable marker, allowing that clone to grow in an appropriate selection medium. It is likely that different sizes of deletions can be generated from a given insertion, and these will be mapped by PCR, Southern blotting, and fluorescent in situ hybridization (FISH) using a series of markers around the insertion site. ES cells containing each deletion can be introduced into mouse blastocysts to make chimeric mice. Matings to created homozygous deletions and compound heterozygous will generate a series of deficiencies for a particular chromosomal region. Mice containing these deletions will be excellent tools for studying the t complex, and this general strategy should be useful for any region of mouse genome.
