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


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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Figure 1: Generation of Fancg (Xrcc9) knockout construct.
Figure 2: Targeting of ES cells by intact BACs.
Figure 3: Generation of knockout mice.


  1. 1

    Tsuzuki, T. & Rancourt, D.E. Embryonic stem cell gene targeting using bacteriophage lambda vectors generated by phage-plasmid recombination. Nucleic Acids Res. 26, 988–993 (1998).

    CAS  Article  Google Scholar 

  2. 2

    Yang, X.W., Model, P. & Heintz, N. Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. Nat. Biotechnol. 15, 859–865 (1997).

    CAS  Article  Google Scholar 

  3. 3

    Yu, D. et al. An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. USA 97, 5978–5983 (2000).

    CAS  Article  Google Scholar 

  4. 4

    Zhang, Y., Buchholz, F., Muyrers, J.P. & Stewart, A.F. A new logic for DNA engineering using recombination in Escherichia coli. Nat. Genet. 20, 123–128 (1998).

    CAS  Article  Google Scholar 

  5. 5

    Zhang, P., Li, M.Z. & Elledge, S.J. Towards genetic genome projects: genomic library screening and gene-targeting vector construction in a single step. Nat. Genet. 30, 31–39 (2002).

    Article  Google Scholar 

  6. 6

    Murphy, K.C. Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. J. Bacteriol. 180, 2063–2071 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Angrand, P.O., Daigle, N., van der Hoeven, F., Scholer, H.R. & Stewart, A.F. Simplified generation of targeting constructs using ET recombination. Nucleic Acids Res. 27, e16 (1999).

    CAS  Article  Google Scholar 

  8. 8

    Koller, B.H. & Smithies, O. Altering genes in animals by gene targeting. Annu. Rev. Immunol. 10, 705–730 (1992).

    CAS  Article  Google Scholar 

  9. 9

    Soriano, P. Gene targeting in ES cells. Annu. Rev. Neurosci. 18, 1–18 (1995).

    CAS  Article  Google Scholar 

  10. 10

    Deng, C. & Capecchi, M.R. Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus. Mol. Cell Biol. 12, 3365–3371 (1992).

    CAS  Article  Google Scholar 

  11. 11

    Hasty, P., Rivera-Perez, J. & Bradley, A. The length of homology required for gene targeting in embryonic stem cells. Mol. Cell Biol. 11, 5586–5591 (1991).

    CAS  Article  Google Scholar 

  12. 12

    Poteete, A.R. & Fenton, A.C. Lambda red-dependent growth and recombination of phage P22. Virology 134, 161–167 (1984).

    CAS  Article  Google Scholar 

  13. 13

    El Karoui, M., Amundsen, S.K., Dabert, P. & Gruss, A. Gene replacement with linear DNA in electroporated wild-type Escherichia coli. Nucleic Acids Res. 27, 1296–1299 (1999).

    CAS  Article  Google Scholar 

  14. 14

    Deichmann, M., Bentz, M. & Haas, R. Ultra-sensitive FISH is a useful tool for studying chronic HIV-1 infection. J. Virol. Methods 65, 19–25 (1997).

    CAS  Article  Google Scholar 

  15. 15

    Reid, L.H., Shesely, E.G., Kim, H.S. & Smithies, O. Cotransformation and gene targeting in mouse embryonic stem cells. Mol. Cell Biol. 11, 2769–2777 (1991).

    CAS  Article  Google Scholar 

  16. 16

    Bollag, R.J., Waldman, A.S. & Liskay, R.M. Homologous recombination in mammalian cells. Annu. Rev. Genet. 23, 199–225 (1989).

    CAS  Article  Google Scholar 

  17. 17

    Capecchi, M.R. Altering the genome by homologous recombination. Science 244, 1288–1292 (1989).

    CAS  Article  Google Scholar 

  18. 18

    Thyagarajan, B., McCormick-Graham, M., Romero, D.P. & Campbell, C. Characterization of homologous DNA recombination activity in normal and immortal mammalian cells. Nucleic Acids Res. 24, 4084–4091 (1996).

    CAS  Article  Google Scholar 

  19. 19

    Yang, Y. et al. Targeted disruption of the murine Fanconi anemia gene, Fancg/Xrcc9. Blood 98, 3435–3440 (2001).

    CAS  Article  Google Scholar 

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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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Correspondence to Brian Seed.

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Yang, Y., Seed, B. Site-specific gene targeting in mouse embryonic stem cells with intact bacterial artificial chromosomes. Nat Biotechnol 21, 447–451 (2003).

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