MCB
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Donoho, G.
Right arrow Articles by Berg, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Donoho, G.
Right arrow Articles by Berg, P.

 Previous Article  |  Next Article 

Mol Cell Biol, July 1998, p. 4070-4078, Vol. 18, No. 7
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Analysis of Gene Targeting and Intrachromosomal Homologous Recombination Stimulated by Genomic Double-Strand Breaks in Mouse Embryonic Stem Cells

Greg Donoho,1 dagger Maria Jasin,2 and Paul Berg1 *

Department of Biochemistry, Beckman Center for Molecular and Genetic Medicine, Stanford University Medical School, Stanford, California 94305,1 and Cell Biology and Genetics Program, Sloan-Kettering Institute and Cornell University Graduate School of Medical Sciences, New York, New York 100212

Received 25 February 1998/Returned for modification 27 March 1998/Accepted 28 April 1998

To investigate the effects of in vivo genomic DNA double-strand breaks on the efficiency and mechanisms of gene targeting in mouse embryonic stem cells, we have used a series of insertion and replacement vectors carrying two, one, or no genomic sites for the rare-cutting endonuclease I-SceI. These vectors were introduced into the hypoxanthine phosphoribosyltransferase (hprt) gene to produce substrates for gene-targeting (plasmid-to-chromosome) or intrachromosomal (direct repeat) homologous recombination. Recombination at the hprt locus is markedly increased following transfection with an I-SceI expression plasmid and a homologous donor plasmid (if needed). The frequency of gene targeting in clones with an I-SceI site attains a value of 1%, 5,000-fold higher than that in clones with no I-SceI site. The use of silent restriction site polymorphisms indicates that the frequencies with which donor plasmid sequences replace the target chromosomal sequences decrease with distance from the genomic break site. The frequency of intrachromosomal recombination reaches a value of 3.1%, 120-fold higher than background spontaneous recombination. Because palindromic insertions were used as polymorphic markers, a significant number of recombinants exhibit distinct genotypic sectoring among daughter cells from a single clone, suggesting the existence of heteroduplex DNA in the original recombination product.

* Corresponding author. Mailing address: Department of Biochemistry, Beckman Center for Molecular and Genetic Medicine, Stanford University Medical School, Stanford, CA 94305. Phone: (650) 723-6170. Fax: (650) 725-4951. E-mail: pberg{at}cmgm.stanford.edu.

dagger Present address: Lexicon Genetics, Inc., The Woodlands, TX 77381.

Mol Cell Biol, July 1998, p. 4070-4078, Vol. 18, No. 7
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:



Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
J. Bacteriol. J. Virol. Eukaryot. Cell
Microbiol. Mol. Biol. Rev. Clin. Vaccine Immunol. All ASM Journals

Copyright © 1998 by the American Society for Microbiology. All rights reserved.