Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae

The mitochondrial intron-encoded endonuclease I-SceI of Saccharomyces cerevisiae has an 18-bp recognition sequence and, therefore, has a very low probability of cutting DNA, even within large genomes. We demonstrate that double-strand breaks can be initiated by the I-SceI endonuclease at a predetermined location in the mouse genome and that the breaks can be repaired with a donor molecule homologous regions flanking the breaks. This induced homologous recombination is approximately 2 orders of magnitude more frequent than spontaneous homologous recombination and at least 10 times more frequent than random integration near an active promoter. As a consequence of induced homologous recombination, a heterologous novel sequence can be inserted at the site of the break. This recombination can occur at a variety of chromosomal targets in differentiated and multipotential cells. These results demonstrate homologous recombination involving chromosomal DNA by the double-strand break repair mechanism in mammals and show the usefulness of very rare cutter endonucleases, such as I-SceI, for designing genome rearrangements.

[1]  R. Bronson,et al.  Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene , 1991, Cell.

[2]  R. Jessberger,et al.  Repair of deletions and double-strand gaps by homologous recombination in a mammalian in vitro system , 1991, Molecular and cellular biology.

[3]  J. Murray,et al.  Site-specific recombinases: tools for genome engineering. , 1993, Trends in genetics : TIG.

[4]  R. Kucherlapati,et al.  Insertion of DNA sequences into the human chromosomal β-globin locus by homologous recombination , 1985, Nature.

[5]  P. Rouet,et al.  Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[6]  N. Henderson,et al.  Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[7]  A. Bernstein,et al.  RNA tumor viruses , 1982 .

[8]  C. Bonnerot,et al.  Application of LacZ gene fusions to postimplantation development. , 1993, Methods in enzymology.

[9]  Mario R. Capecchi,et al.  Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes , 1988, Nature.

[10]  G. Schmitz,et al.  Cleavage of yeast and bacteriophage T7 genomes at a single site using the rare cutter endonuclease I-Sce I , 1991, Nucleic Acids Res..

[11]  N. Sternberg,et al.  Intermolecular recombination between DNAs introduced into mouse L cells is mediated by a nonconservative pathway that leads to crossover products , 1990, Molecular and cellular biology.

[12]  N. Sternberg,et al.  Repair of double-stranded DNA breaks by homologous DNA fragments during transfer of DNA into mouse L cells , 1990, Molecular and cellular biology.

[13]  D. Brenner,et al.  Double-strand gap repair results in homologous recombination in mouse L cells. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[14]  D. Baltimore,et al.  Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus , 1983, Cell.

[15]  M. Seidman.  Intermolecular homologous recombination between transfected sequences in mammalian cells is primarily nonconservative , 1987, Molecular and cellular biology.

[16]  A. Gatignol,et al.  Phleomycin resistance as a dominant selectable marker in CHO cells , 1988, Somatic cell and molecular genetics.

[17]  C. Bonnerot,et al.  Capture of a cellular transcriptional unit by a retrovirus: mode of provirus activation in embryonal carcinoma cells , 1992, Journal of virology.

[18]  B. Dujon,et al.  Site-specific recombination determined by I-SceI, a mitochondrial group I intron-encoded endonuclease expressed in the yeast nucleus. , 1992, Genetics.

[19]  Jack W. Szostak,et al.  The double-strand-break repair model for recombination , 1983, Cell.

[20]  Bernard Dujon,et al.  An intron-encoded protein is active in a gene conversion process that spreads an intron into a mitochondrial gene , 1985, Cell.

[21]  M. Shulman,et al.  Gene replacement with one-sided homologous recombination , 1992, Molecular and cellular biology.

[22]  J. Nicolas,et al.  [6] Mouse teratocarcinoma cells , 1987 .

[23]  P. Briand,et al.  Application of LacZ gene fusions to postimplantation development. , 1993 .