To investigate the effects of in vivo genomic DNA double-strand breaks on the efficiency and mechanisms of gene targeting in mouse embryonic stem cells, we have used a series of insertion and replacement vectors carrying two, one, or no genomic sites for the rare-cutting endonuclease I-SceI. These vectors were introduced into the hypoxanthine phosphoribosyltransferase (hprt) gene to produce substrates for gene-targeting (plasmid-to-chromosome) or intrachromosomal (direct repeat) homologous recombination. Recombination at the hprt locus is markedly increased following transfection with an I-SceI expression plasmid and a homologous donor plasmid (if needed). The frequency of gene targeting in clones with an I-SceI site attains a value of 1%, 5,000-fold higher than that in clones with no I-SceI site. The use of silent restriction site polymorphisms indicates that the frequencies with which donor plasmid sequences replace the target chromosomal sequences decrease with distance from the genomic break site. The frequency of intrachromosomal recombination reaches a value of 3.1%, 120-fold higher than background spontaneous recombination. Because palindromic insertions were used as polymorphic markers, a significant number of recombinants exhibit distinct genotypic sectoring among daughter cells from a single clone, suggesting the existence of heteroduplex DNA in the original recombination product.The ability to generate transgenic animals with predictable genomic alterations has opened the way for analyzing the physiological consequences of these changes in the whole animal. A significant obstacle to obtaining transgenic animals with targeted modifications of their genomes is the fact that homologous recombination in mouse embryonic stem (ES) cells is relatively infrequent (10 Ϫ5 to 10 Ϫ8 events per transfected cell) (8,15,24). Two complementary strategies have been used to circumvent this limitation: one relies on the use of positive selectable markers in the targeting DNA to identify transformants (18,37,38); the second relies on positive-negative selection strategies, i.e., selection strategies that eliminate cells that have undergone nontargeted events (24). Such positivenegative selection procedures have been adopted to eliminate the nonhomologous events, which are far more frequent (perhaps 10-to 10 5 -fold) than the desired homologous recombination (23,37,38,42). In each instance, however, putative targeted transformants must be screened by restriction analysis to confirm that the desired change has been made.Rather than focusing on improved means for selection of the desired events, we have sought to overcome the difficulties stemming from the low yield of accurately targeted events by developing a way to increase the frequency of homologous targeted events. Double-strand breaks (DSBs) made by rarecutting endonucleases have been shown to stimulate mitotic homologous recombination in yeast (reviewed in reference 13), plant (29), and mammalian cells (4,6,17,33,35). Recombinational repair of DSBs relies on the abi...

The Brca2 tumor-suppressor gene contributes to genomic stability, at least in part by a role in homologous recombinational repair. BRCA2 protein is presumed to function in homologous recombination through interactions with RAD51. Both exons 11 and 27 of Brca2 code for domains that interact with RAD51; exon 11 encodes eight BRC motifs, whereas exon 27 encodes a single, distinct interaction domain. Deletion of all RAD51-interacting domains causes embryonic lethality in mice. A less severe phenotype is seen with BRAC2 truncations that preserve some, but not all, of the BRC motifs. These mice can survive beyond weaning, but are runted and infertile, and die very young from cancer. Cells from such mice show hypersensitivity to some genotoxic agents and chromosomal instability. Here, we have analyzed mice and cells with a deletion of only the RAD51-interacting region encoded by exon 27. Mice homozygous for this mutation (called brca2(lex1)) have a shorter life span than that of control littermates, possibly because of early onsets of cancer and sepsis. No other phenotype was observed in these animals; therefore, the brca2(lex1) mutation is less severe than truncations that delete some BRC motifs. However, at the cellular level, the brca2(lex1) mutation causes reduced viability, hypersensitivity to the DNA interstrand crosslinking agent mitomycin C, and gross chromosomal instability, much like more severe truncations. Thus, the extreme carboxy-terminal region encoded by exon 27 is important for BRCA2 function, probably because it is required for a fully functional interaction between BRCA2 and RAD51.

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We have constructed and characterized a polyoma virus-based plasmid that is maintained as an autonomously replicating extrachromosomal element (episome) in mouse embryonic stem (ES) cells. Plasmid pMGD20neo contains the polyoma origin of replication harboring a mutated enhancer (PyFlO), a modified polyoma early region that encodes the large tumor (T) antigen only, and a gene that confers resistance to G418 (neo). After transfection, the plasmid replicates in ES cells and is maintained as an extrachromosomal element in 15% of G418-resistant clones. Integration of the plasmid DNA is undetectable for at least 28 cell generations. In one clone, the transfected DNA persists unaltered as an episome at 10-30 copies per cell for at least 74 cell generations in the presence of G418. Cells that maintain the autonomously replicating plasmid can efficiently replicate and maintain a second plasmid that carries the polyoma origin of replication. Polyoma virus DNA replicates as free, unintegrated minichromosomes in infected mouse cells (6). Three related proteins, encoded by the polyoma virus early region, are expressed shortly after infection: large tumor (T) antigen, middle T, and small T. These proteins are translated from three different mRNAs generated by alternative splicing of the primary transcript (7). Large T is required for initiating viral DNA replication (8, 9), while middle T is responsible for the viral transforming activity (10), and small T is suspected of having a subsidiary role in DNA replication (11) and/or in transformation (12, 13). Polyoma virus mutants whose DNA lacks the entire large T intron (14) or where the splice sites used in the formation of the properly processed middle T and small T

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