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Targeted homologous recombination at the endogenous adenine phosphoribosyltransferase locus in Chinese hamster cells.

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Affiliations

  • 1. University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville 78957.
      Authors
    • Adair GM 1
    (1 author)

Proceedings of the National Academy of Sciences of the United States of America, 01 Jun 1989, 86(12):4574-4578
https://doi.org/10.1073/pnas.86.12.4574 PMID: 2734308 PMCID: PMC287313

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Abstract 

We have developed a system that permits analysis of targeted homologous recombination at an endogenous, chromosomal gene locus in cultured mammalian cells. Using a hemizygous, adenine phosphoribosyltransferase (APRT)-deficient, Chinese hamster ovary (CHO) cell mutant as a transfection recipient, we have demonstrated correction of a nonrevertible deletion mutation by targeted homologous recombination. Transfection with a plasmid carrying a fragment of the APRT gene yielded APRT+ recombinants at a frequency of approximately 4.1 x 10(-7). The ratio of targeted recombination to nontargeted integrations of plasmid sequences was approximately 1:4000. Analysis of 31 independent APRT+ recombinants revealed conversions of the endogenous APRT gene, targeted integration at the APRT locus, and a third class of events in which the plasmid donor APRT fragment was converted to a full-length, functional gene.

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Proc Natl Acad Sci U S A. 1989 Jun; 86(12): 4574–4578.
PMCID: PMC287313
PMID: 2734308

Targeted homologous recombination at the endogenous adenine phosphoribosyltransferase locus in Chinese hamster cells.

G M Adair, R S Nairn, J H Wilson, M M Seidman, K A Brotherman, C MacKinnon, and J B Scheerer
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Abstract

We have developed a system that permits analysis of targeted homologous recombination at an endogenous, chromosomal gene locus in cultured mammalian cells. Using a hemizygous, adenine phosphoribosyltransferase (APRT)-deficient, Chinese hamster ovary (CHO) cell mutant as a transfection recipient, we have demonstrated correction of a nonrevertible deletion mutation by targeted homologous recombination. Transfection with a plasmid carrying a fragment of the APRT gene yielded APRT+ recombinants at a frequency of approximately 4.1 x 10(-7). The ratio of targeted recombination to nontargeted integrations of plasmid sequences was approximately 1:4000. Analysis of 31 independent APRT+ recombinants revealed conversions of the endogenous APRT gene, targeted integration at the APRT locus, and a third class of events in which the plasmid donor APRT fragment was converted to a full-length, functional gene.

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Selected References

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