Double-strand breaks at the target locus stimulate gene targeting in embryonic stem cells

Nucleic Acids Res. 1995 Dec 25;23(24):5012-9. doi: 10.1093/nar/23.24.5012.


Double-strand breaks (DSBs) are recombinogenic lesions in chromosomal DNA in yeast, Drosophila and Caenorhabditis elegans. Recent studies in mammalian cells utilizing the I-Scel endonuclease have demonstrated that in some immortalized cell lines DSBs in chromosomal DNA are also recombinogenic. 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 I-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-Scel 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 'two-sided', 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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • Cell Line
  • DNA / genetics*
  • DNA Damage*
  • Deoxyribonucleases, Type II Site-Specific / biosynthesis
  • Deoxyribonucleases, Type II Site-Specific / genetics
  • Gene Transfer Techniques
  • Kanamycin Kinase
  • Mice
  • Molecular Sequence Data
  • Phosphotransferases (Alcohol Group Acceptor) / genetics*
  • Recombination, Genetic*
  • Saccharomyces cerevisiae Proteins
  • Stem Cells*


  • Saccharomyces cerevisiae Proteins
  • DNA
  • Phosphotransferases (Alcohol Group Acceptor)
  • Kanamycin Kinase
  • SCEI protein, S cerevisiae
  • Deoxyribonucleases, Type II Site-Specific