INTRODUCTION OF DOUBLE-STRAND BREAKS INTO THE GENOME OF MOUSE CELLS BY EXPRESSION OF A RARE-CUTTING ENDONUCLEASE

To maintain genomic integrity, double-strand breaks (DSBs) in chromoso mal DNA must be repaired. In mammalian systems, the analysis of the re pair of chromosomal DSBs has been limited by the inability to introduc e well-defined DSBs in genomic DNA. In this study, we created specific DSBs in mouse chromosomes for the first time, using an expression sys tem for a rare-cutting endonuclease, I-SceI. A genetic assay has been devised to monitor the repair of DSBs, whereby cleavage sites for I-Sc eI have been integrated into the mouse genome in two tandem neomycin p hosphotransferase genes. We find that cleavage of the I-SceI sites is very efficient, with at least 12% of stably transfected cells having a t least one cleavage event and, of these, more than 70% have undergone cleavage at both I-SceI sites. Cleavage of both sites in a fraction o f clones deletes 3.8 kb of intervening chromosomal sequences. We find that the DSBs are repaired by both homologous and nonhomologous mechan isms. Nonhomologous repair events frequently result in small deletions after rejoining of the two DNA ends. Some of these appear to occur by simple blunt-ended ligation, whereas several others may occur through annealing of short regions of terminal homology. The DSBs are apparen tly recombinogenic, stimulating gene targeting of a homologous fragmen t by more than 2 orders of magnitude. Whereas gene-targeted clones are nearly undetectable without endonuclease expression, they represent a pproximately 10% of cells transfected with the I-SceI expression vecto r. Gene targeted clones are of two major types, those that occur by tw o-sided homologous recombination with the homologous fragment and thos e that occur by one-sided homologous recombination. Our results are ex pected to impact a number of areas in the study of mammalian genome dy namics, including the analysis of the repair of DSBs and homologous re combination and, potentially, molecular genetic analyses of mammalian genomes.