INDUCTION OF HOMOLOGOUS RECOMBINATION IN MAMMALIAN CHROMOSOMES BY USING THE I-SCEI SYSTEM OF SACCHAROMYCES-CEREVISIAE

The mitochondrial intron-encoded endonuclease I-SeeI of Saccharomgces cervisiae has an 18-bp recognition sequence and, therefore, has a very low probability of cutting DNA, even within large genomes. We demonst rate that double-strand breaks can be initiated by the I-SeeI endonucl ease at a predetermined location in the mouse genome and that the brea ks can be repaired with a donor molecule homologous with regions flank ing the breaks. This induced homologous recombination is approximately 2 orders of magnitude more frequent than spontaneous homologous recom bination and at least 10 times more frequent than random integration n ear an active promoter. As a consequence of induced homologous recombi nation, a heterologous novel sequence can be inserted at the site of t he break. This recombination can occur at a variety of chromosomal tar gets in differentiated and multipotential cells. These results demonst rate homologous recombination involving chromosomal DNA by the double- strand break repair mechanism in mammals and show the usefulness of ve ry rare cutter endonucleases, such as I-SeeI, for designing genome rea rrangements.