| Abstract: |
Double-strand breaks (DSBs) are recomblnogenlc lesions In chromosomal DNA in yeast, Drosophlla and Caenortiabdltls elegans. Recent studies In mammalian cells utilizing the l-Scel endonuclease have demonstrated that in some immortalized cell lines DSBs In chromosomal DNA are also recomblnogenic. We have now tested embryonic stem (ES) cells, a non-transformed mouse cell line frequently used in gene targeting studies. We find that a DSB introduced by l-Scel stimulates gene targeting at a selectable neo locus at least 50-fold. The enhanced level of targeting is achieved by transient expression of the I-Sce) endonuclease. In 97% of targeted clones a single base pair polymorphism in the transfected homologous fragment was incorporated into the target locus. Analysis of the targeted locus demonstrated that most of the homologous recombination events were 'twosided', in contrast to previous studies In 3T3 cells in which 'one-sided' homologous events predominated. Thus ES cells may be more faithful in incorporating homologous fragments into their genome than other cells in culture. © 1995 Oxford University Press. |
