Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells Journal Article


Authors: Rouet, P.; Smih, F.; Jasin, M.
Article Title: Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells
Abstract: Double-strand breaks introduced into DNA in vivo have been shown to enhance homologous recombination in a variety of chromosomal and extrachromosomal loci in Saccharomyces cerevisiae. To introduce double- strand breaks in DNA at defined locations in mammalian cells, we have constructed a mammalian expression vector for a modified form of I-Sce I, a yeast mitochondrial intron-encoded endonuclease with an 18-bp recognition sequence. Expression of the modified I-Sce I endonuclease in COS1 cells results in cleavage of model recombination substrates and enhanced extrachromosomal recombination, as assayed by chloramphenicol acetyltransferase activity and Southern blot analysis. Constitutive expression of the endonuclease in mouse 3T3 cells is not lethal, possibly due to either the lack of I-Sce I sites in the genome or sufficient repair of them. Expression of an endonuclease with such a long recognition sequence will provide a powerful approach to studying a number of molecular processes in mammalian cells, including homologous recombination.
Keywords: gene sequence; nonhuman; animal cell; mouse; animal; mice; dna repair; cell line; gene locus; transfection; cercopithecus aethiops; dna strand breakage; kidney; dna; genetic recombination; molecular sequence data; recombination, genetic; mammal; escherichia coli; base sequence; plasmids; binding sites; endonuclease; 3t3 cells; oligodeoxyribonucleotides; restriction mapping; mouse cells; promoter regions (genetics); deoxyribonucleases, type ii site-specific; blotting, southern; priority journal; article; dna restriction enzymes; chloramphenicol o-acetyltransferase; support, non-u.s. gov't; support, u.s. gov't, p.h.s.; sarcoma viruses, avian; extrachromosomal recombination; i-sce i
Journal Title: Proceedings of the National Academy of Sciences of the United States of America
Volume: 91
Issue: 13
ISSN: 0027-8424
Publisher: National Academy of Sciences  
Date Published: 1994-06-21
Start Page: 6064
End Page: 6068
Language: English
DOI: 10.1073/pnas.91.13.6064
PROVIDER: scopus
PMCID: PMC44138
PUBMED: 8016116
DOI/URL:
Notes: Export Date: 14 January 2019 -- Article -- CODEN: PNASA C2 -- Source: Scopus
Altmetric
Citation Impact
MSK Authors
  1. Maria Jasin
    246 Jasin