TARGETED DISRUPTION OF THE MOUSE TOPOISOMERASE-I GENE BY CAMPTOTHECINSELECTION

Topoisomerase I has ubiquitous roles in important cellular functions s uch as replication, transcription, and recombination. In order to furt her characterize this enzyme in vivo, we have used gene targeting to i nactivate the mouse Top-I gene. A selection protocol using the topoiso merase I inhibitor camptothecin facilitated isolation of embryonic ste m cell clones containing an inactivated allele; isolation of correctly targeted clones was enhanced 75-fold over that achieved by normal sel ection procedures, The disrupted Top-I allele is embryonic lethal when homozygous, and development of such embryos fails between the 4- and 16-cell stages, Both sperm and oocytes containing the inactive allele maintain viability through the fertilization point, and thus gene expr ession of topoisomerase I is not required for gamete, viability. These studies demonstrate that topoisomerase I is essential for cell growth and division in vivo. The Top-I gene was also shown to be linked to t he agouti locus.