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Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles


The mouse is the leading vertebrate model because its genome can be altered by both random transgenesis and homologous recombination with targeting constructs. Both approaches have been hindered by the size and site limitations implicit in conventional Escherichia coli DNA-engineering methods. Homologous recombination in E. coli, or 'recombineering', has overcome these limitations for bacterial artificial chromosome (BAC) transgenesis1,2,3. Here we applied Red/ET recombineering (using the lambda Redα/Redβ recombinase pair)4,5,6 to generate a 64 kilobase targeting construct that carried two selectable cassettes permitting the simultaneous mutation of the target gene, Mll, at sites 43 kb apart in one round of mouse embryonic stem (ES) cell targeting. The targeting frequency after dual selection was 6%. Because the two selectable cassettes were flanked by FRT or loxP sites, three more alleles can be generated by site-specific recombination. Our approach represents a simple way to introduce changes at two or more sites in a genetic locus, and thereafter generate allele combinations. The size of BAC templates offers new freedom for the design of targeting constructs. Combined with the use of two selectable cassettes placed far apart, BAC-based targeting constructs may be applicable to tasks such as regional exchanges, deletions, and insertions.

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Figure 1: Red/ET engineering of a BAC targeting construct.
Figure 2: Analysis of the allele in ES cells and mice.
Figure 3: Schematic representation of multi-purpose alleles.


  1. 1

    Muyrers, J.P., Zhang, Y. & Stewart, A.F. Techniques: recombinogenic engineering—new options for cloning and manipulating DNA. Trends Biochem. Sci. 26, 325–331 (2001).

    CAS  Article  Google Scholar 

  2. 2

    Copeland, N.G., Jenkins, N.A. & Court, D.L. Recombineering: a powerful new tool for mouse functional genomics. Nat. Rev. Genet. 2, 769–779 (2001).

    CAS  Article  Google Scholar 

  3. 3

    Heintz, N. BAC to the future: the use of bac transgenic mice for neuroscience research. Nat. Rev. Neurosci. 2, 861–870 (2001).

    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

    Muyrers, J.P., Zhang, Y., Testa, G. & Stewart, A.F. Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res. 27, 1555–1557 (1999).

    CAS  Article  Google Scholar 

  6. 6

    Zhang, Y., Muyrers, J.P., Testa, G. & Stewart, A.F. DNA cloning by homologous recombination in Escherichia coli. Nat. Biotechnol. 18, 1314–1317 (2000).

    CAS  Article  Google Scholar 

  7. 7

    Testa, G. & Stewart, A.F. Creating a translocation. Engineering interchromosomal translocations in the mouse. EMBO Rep. 1, 120–121 (2000).

    CAS  Article  Google Scholar 

  8. 8

    Rowley, J.D. The critical role of chromosome translocations in human leukemias. Annu. Rev. Genet. 32, 495–519 (1998).

    CAS  Article  Google Scholar 

  9. 9

    Nilson, I. et al. Exon/intron structure of the human ALL-1 (MLL) gene involved in translocations to chromosomal region 11q23 and acute leukaemias. Br. J. Haematol. 93, 966–972 (1996).

    CAS  Article  Google Scholar 

  10. 10

    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 

  11. 11

    Rigaut, G. et al. A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 17, 1030–1032 (1999).

    CAS  Article  Google Scholar 

  12. 12

    Buchholz, F., Angrand, P.O. & Stewart, A.F. Improved properties of FLP recombinase evolved by cycling mutagenesis. Nat. Biotechnol. 16, 657–662 (1998).

    CAS  Article  Google Scholar 

  13. 13

    Zambrowicz, B.P. et al. Disruption of overlapping transcripts in the ROSA βgeo26 gene trap strain leads to widespread expression of β-galactosidase in mouse embryos and hematopoietic cells. Proc. Natl. Acad. Sci. USA 94, 3789–3794 (1997).

    CAS  Article  Google Scholar 

  14. 14

    Moens, C.B., Auerbach, A.B., Conlon, R.A., Joyner, A.L. & Rossant, J. A targeted mutation reveals a role for N-myc in branching morphogenesis in the embryonic mouse lung. Genes Dev. 6, 691–704 (1992).

    CAS  Article  Google Scholar 

  15. 15

    Muyrers, J.P. et al. Point mutation of bacterial artificial chromosomes by ET recombination. EMBO Rep. 1, 239–243 (2000).

    CAS  Article  Google Scholar 

  16. 16

    Ellis, H.M., Yu, D., DiTizio, T. & Court, D.L. High-efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc. Natl. Acad. Sci. USA 98, 6742–6746 (2001).

    CAS  Article  Google Scholar 

  17. 17

    Baer, A. & Bode, J. Coping with kinetic and thermodynamic barriers: RMCE, an efficient strategy for the targeted integration of transgenes. Curr. Opin. Biotechnol. 12, 473–480 (2001).

    CAS  Article  Google Scholar 

  18. 18

    Meyers, E.N., Lewandoski, M. & Martin, G.R. An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination. Nat. Genet. 18, 136–141 (1998).

    CAS  Article  Google Scholar 

  19. 19

    Nagy, A. et al. Dissecting the role of N-myc in development using a single targeting vector to generate a series of alleles. Curr. Biol. 8, 661–664 (1998).

    CAS  Article  Google Scholar 

  20. 20

    Andreas, S., Schwenk, F., Kuter-Luks, B., Faust, N. & Kuhn, R. Enhanced efficiency through nuclear localization signal fusion on phage PhiC31-integrase: activity comparison with Cre and FLPe recombinase in mammalian cells. Nucleic Acids Res. 30, 2299–2306 (2002).

    CAS  Article  Google Scholar 

  21. 21

    Buchholz, F. & Stewart, A.F. Alteration of Cre recombinase site specificity by substrate-linked protein evolution. Nat. Biotechnol. 19, 1047–1052 (2001).

    CAS  Article  Google Scholar 

  22. 22

    Ayton, P. et al. Truncation of the Mll gene in exon 5 by gene targeting leads to early preimplantation lethality of homozygous embryos. Genesis 30, 201–212 (2001).

    CAS  Article  Google Scholar 

  23. 23

    Benes, V., Kilger, C., Voss, H., Paabo, S. & Ansorge, W. Direct primer walking on P1 plasmid DNA. Biotechniques 23, 98–100 (1997).

    CAS  Article  Google Scholar 

  24. 24

    Nichols, J., Evans, E.P. & Smith, A.G. Establishment of germ-line-competent embryonic stem (ES) cells using differentiation-inhibiting activity. Development 110, 1341–1348 (1990).

    CAS  Google Scholar 

  25. 25

    Joyner, A.L. (ed.). Gene Targeting. A Practical Approach (Oxford University Press, New York, 2000).

    Google Scholar 

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We wish to thank Konstantinos Anastassiadis, William Brown, Frank van der Hoeven, Robin Lovell-Badge, and Daniela Nebenius-Oosthuizen for discussions and help. This work was partly funded by a grant from the Volkswagen Foundation, Program on Conditional Mutagenesis. The work was initiated at the European Molecular Biology Laboratory (EMBL), Heidelberg.

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Correspondence to A. Francis Stewart.

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Competing interests

The Red/ET DNA engineering technology is the subject of two issued patents authored by Drs. Zhang and Stewart. These patents are owned by EMBL which issued exclusive rights to GeneBridges GmbH, a company cofounded by Drs. Zhang and Stewart to develop the commercial implications of the patents. Dr. Zhang is CSO and Dr. Stewart chairperson of GeneBridges GmbH. Both are shareholders.

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Testa, G., Zhang, Y., Vintersten, K. et al. Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles. Nat Biotechnol 21, 443–447 (2003).

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