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Site-specific gene targeting in mouse embryonic stem cells with intact bacterial artificial chromosomes

Nature Biotechnology volume 21, pages 447451 (2003) | Download Citation

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Abstract

Homologous recombination in Escherichia coli simplifies the generation of gene targeting constructs for transduction into mouse embryonic stem (ES) cells1,2,3,4,5,6,7. Taking advantage of the extensive homology provided by intact bacterial artificial chromosomes (BACs), we have developed an efficient method for preparing targeted gene disruptions in ES cells. Correctly integrated clones were identified by a simple screening procedure based on chromosomal fluorescence in situ hybridization (FISH). To date, five mutant lines have been generated and bred to homozygosity by this approach.

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Acknowledgements

We thank Naifang Lu and Jeannie T. Lee for help in developing the FISH protocol, Naifang Lu for blastocyst injections, and Vidya Kunjathoor, Yanhong Ma, and Amy Stirman for assistance. This work was supported by grants from the US National Institutes of Health (AI27849, AI46731, and HL66678 to B.S.) and a postdoctoral fellowship from the Cancer Research Institute (to Y.Y.).

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Affiliations

  1. Department of Molecular Biology, Massachusetts General Hospital, and Department of Genetics, Harvard Medical School, Boston, MA 02114.

    • Yi Yang
    •  & Brian Seed

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The authors declare no competing financial interests.

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Correspondence to Brian Seed.

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DOI

https://doi.org/10.1038/nbt803

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