Double-strand breaks at the target locus stimulate gene targeting in embryonic stem cells.
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.
Smih
F
F
Cell Biology and Genetics Program, Sloan-Kettering Institute, New York, NY 10021, USA.
Rouet
P
P
Romanienko
P J
PJ
Jasin
M
M
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000235:genetics*; D004249:DNA Damage; D015252:Deoxyribonucleases, Type II Site-Specific / Q000096:biosynthesis / Q000235:genetics; D018014:Gene Transfer Techniques; D019868:Kanamycin Kinase; D051379:Mice; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics*; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins; D013234:Stem Cells
D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific