Endonuclease-induced, targeted homologous extrachromosomal recombination in Xenopus oocytes

David Segal, Dana Carroll

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

Homologous recombination in gene targeting in most organisms occurs by an inefficient mechanism. Inducing a double-strand break in the chromosomal target may increase this efficiency by allowing recombination to proceed by the highly efficient single-strand annealing mechanism. A gene targeting experiment was modeled in Xenopus oocytes by using a circular plasmid to mimic the chromosomal target site and a homologous linear molecule (pick-up fragment or PUF) as an analogue of the vector DNA. When those two molecules were simply injected together, no recombination was observed. In contrast, when the circular plasmid was cleaved in vivo by injection of the site- specific endonuclease, I-Sce I, relatively efficient intermolecular recombination occurred, involving up to 17% of the cleaved molecules. Recombination was dependent on the stability of the PUF; product yield was increased by using longer fragments and by injecting larger amounts of linear DNA. both of which increased the lifetime of the PUF in the oocytes. These results demonstrate that in vivo double-strand breaks can induce homologous recombination of reluctant substrates and may be useful in augmenting the efficiency of gene targeting.

Original languageEnglish (US)
Pages (from-to)806-810
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume92
Issue number3
DOIs
StatePublished - Jan 31 1995
Externally publishedYes

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Endonucleases
Homologous Recombination
Xenopus
Genetic Recombination
Oocytes
Gene Targeting
Plasmids
Chromosome Breakage
Deoxyribonuclease I
DNA
Injections

Keywords

  • gene targeting
  • I-Sce I
  • oocyte injection
  • single-strand annealing

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

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abstract = "Homologous recombination in gene targeting in most organisms occurs by an inefficient mechanism. Inducing a double-strand break in the chromosomal target may increase this efficiency by allowing recombination to proceed by the highly efficient single-strand annealing mechanism. A gene targeting experiment was modeled in Xenopus oocytes by using a circular plasmid to mimic the chromosomal target site and a homologous linear molecule (pick-up fragment or PUF) as an analogue of the vector DNA. When those two molecules were simply injected together, no recombination was observed. In contrast, when the circular plasmid was cleaved in vivo by injection of the site- specific endonuclease, I-Sce I, relatively efficient intermolecular recombination occurred, involving up to 17{\%} of the cleaved molecules. Recombination was dependent on the stability of the PUF; product yield was increased by using longer fragments and by injecting larger amounts of linear DNA. both of which increased the lifetime of the PUF in the oocytes. These results demonstrate that in vivo double-strand breaks can induce homologous recombination of reluctant substrates and may be useful in augmenting the efficiency of gene targeting.",
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AU - Segal, David

AU - Carroll, Dana

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N2 - Homologous recombination in gene targeting in most organisms occurs by an inefficient mechanism. Inducing a double-strand break in the chromosomal target may increase this efficiency by allowing recombination to proceed by the highly efficient single-strand annealing mechanism. A gene targeting experiment was modeled in Xenopus oocytes by using a circular plasmid to mimic the chromosomal target site and a homologous linear molecule (pick-up fragment or PUF) as an analogue of the vector DNA. When those two molecules were simply injected together, no recombination was observed. In contrast, when the circular plasmid was cleaved in vivo by injection of the site- specific endonuclease, I-Sce I, relatively efficient intermolecular recombination occurred, involving up to 17% of the cleaved molecules. Recombination was dependent on the stability of the PUF; product yield was increased by using longer fragments and by injecting larger amounts of linear DNA. both of which increased the lifetime of the PUF in the oocytes. These results demonstrate that in vivo double-strand breaks can induce homologous recombination of reluctant substrates and may be useful in augmenting the efficiency of gene targeting.

AB - Homologous recombination in gene targeting in most organisms occurs by an inefficient mechanism. Inducing a double-strand break in the chromosomal target may increase this efficiency by allowing recombination to proceed by the highly efficient single-strand annealing mechanism. A gene targeting experiment was modeled in Xenopus oocytes by using a circular plasmid to mimic the chromosomal target site and a homologous linear molecule (pick-up fragment or PUF) as an analogue of the vector DNA. When those two molecules were simply injected together, no recombination was observed. In contrast, when the circular plasmid was cleaved in vivo by injection of the site- specific endonuclease, I-Sce I, relatively efficient intermolecular recombination occurred, involving up to 17% of the cleaved molecules. Recombination was dependent on the stability of the PUF; product yield was increased by using longer fragments and by injecting larger amounts of linear DNA. both of which increased the lifetime of the PUF in the oocytes. These results demonstrate that in vivo double-strand breaks can induce homologous recombination of reluctant substrates and may be useful in augmenting the efficiency of gene targeting.

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