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High-efficiency counterselection recombineering for site-directed mutagenesis in bacterial artificial chromosomes

Nature Methods volume 9, pages 103–109 (2012)Cite this article

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Abstract

Whereas bacterial artificial chromosomes (BACs) offer many advantages in studies of gene and protein function, generation of seamless, precisely mutated BACs has been difficult. Here we describe a counterselection-based recombineering method and its accompanying reagents. After identifying intramolecular recombination as the major problem in counterselection, we built a strategy to reduce these unwanted events by expressing Redβ alone at the crucial step. We enhanced this method by using phosphothioated oligonucleotides, using a sequence-altered rpsL counterselection gene and developing online software for oligonucleotide design. We illustrated this method by generating transgenic mammalian cell lines carrying small interfering RNA–resistant and point-mutated BAC transgenes. Using this approach, we generated mutated TACC3 transgenes to identify phosphorylation-specific spindle defects after knockdown of endogenous TACC3 expression. Our results highlight the complementary use of precisely mutated BAC transgenes and RNA interference in the study of cell biology at physiological expression levels and regulation.

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Figure 1: Conventional recombineering counterselection strategy.
Figure 2: Effect of different combinations of Red proteins.
Figure 3: Efficiency of the new counterselection strategy.
Figure 4: RNAi-resistant and phosphorylation-site mutations in TACC3 BAC transgenes allow the identification of subtle phospho-dependent spindle morphology phenotypes.

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References

  1. 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).

    Article  CAS  Google Scholar 

  2. 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).

    Article  CAS  Google Scholar 

  3. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Kittler, R. et al. RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells. Proc. Natl. Acad. Sci. USA 102, 2396–2401 (2005).

    Article  CAS  Google Scholar 

  6. Hofemeister, H. et al. Recombineering, transfection, Western, IP and ChIP methods for protein tagging via gene targeting or BAC transgenesis. Methods 53, 437–452 (2011).

    Article  CAS  Google Scholar 

  7. Sarov, M. et al. A recombineering pipeline for functional genomics applied to Caenorhabditis elegans. Nat. Methods 3, 839–844 (2006).

    Article  CAS  Google Scholar 

  8. Poser, I. et al. BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals. Nat. Methods 5, 409–415 (2008).

    Article  CAS  Google Scholar 

  9. Skarnes, W.C. et al. A conditional knockout resource for the genome-wide study of mouse gene function. Nature 474, 337–342 (2011).

    Article  CAS  Google Scholar 

  10. Bird, A.W. & Hyman, A.A. Building a spindle of the correct length in human cells requires the interaction between TPX2 and Aurora A. J. Cell Biol. 182, 289–300 (2008).

    Article  CAS  Google Scholar 

  11. Hubner, N.C. et al. Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions. J. Cell Biol. 189, 739–754 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  13. Warming, S., Costantino, N., Court, D.L., Jenkins, N.A. & Copeland, N.G. Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res. 33, e36 (2005).

    Article  Google Scholar 

  14. Zhang, Y., Muyrers, J.P., Rientjes, J. & Stewart, A.F. Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cells. BMC Mol. Biol. 4, 1 (2003).

    Article  Google Scholar 

  15. Wang, J. et al. An improved recombineering approach by adding RecA to λ Red recombination. Mol. Biotechnol. 32, 43–53 (2006).

    Article  Google Scholar 

  16. Russell, C.B. & Dahlquist, F.W. Exchange of chromosomal and plasmid alleles in Escherichia coli by selection for loss of a dominant antibiotic sensitivity marker. J. Bacteriol. 171, 2614–2618 (1989).

    Article  CAS  Google Scholar 

  17. Wang, S., Zhao, Y., Leiby, M. & Zhu, J. A new positive/negative selection scheme for precise BAC recombineering. Mol. Biotechnol. 42, 110–116 (2009).

    Article  CAS  Google Scholar 

  18. Westenberg, M., Soedling, H.M., Mann, D.A., Nicholson, L.J. & Dolphin, C.T. Counter-selection recombineering of the baculovirus genome: a strategy for seamless modification of repeat-containing BACs. Nucleic Acids Res. 38, e166 (2010).

    Article  Google Scholar 

  19. Court, R., Cook, N., Saikrishnan, K. & Wigley, D. The crystal structure of λ-Gam protein suggests a model for RecBCD inhibition. J. Mol. Biol. 371, 25–33 (2007).

    Article  CAS  Google Scholar 

  20. Murphy, K.C. The λ Gam protein inhibits RecBCD binding to dsDNA ends. J. Mol. Biol. 371, 19–24 (2007).

    Article  CAS  Google Scholar 

  21. Erler, A. et al. Conformational adaptability of Redβ during DNA annealing and implications for its structural relationship with Rad52. J. Mol. Biol. 391, 586–598 (2009).

    Article  CAS  Google Scholar 

  22. Kovall, R. & Matthews, B.W. Toroidal structure of λ-exonuclease. Science 277, 1824–1827 (1997).

    Article  CAS  Google Scholar 

  23. Wu, Z. et al. Domain structure and DNA binding regions of (protein from bacteriophage λ. J. Biol. Chem. 281, 25205–25214 (2006).

    Article  CAS  Google Scholar 

  24. Maresca, M. et al. Single-stranded heteroduplex intermediates in λ Red homologous recombination. BMC Mol. Biol. 11, 54 (2010).

    Article  Google Scholar 

  25. Mosberg, J.A., Lajoie, M.J. & Church, G.M. λ red recombineering in Escherichia coli occurs through a fully single-stranded intermediate. Genetics 186, 791–799 (2010).

    Article  CAS  Google Scholar 

  26. 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).

    Article  CAS  Google Scholar 

  27. Stahl, M.M. et al. Annealing vs. invasion in phage λ recombination. Genetics 147, 961–977 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Anastassiadis, K. et al. Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice. Dis. Model. Mech. 2, 508–515 (2009).

    Article  CAS  Google Scholar 

  29. Muyrers, J.P., Zhang, Y., Buchholz, F. & Stewart, A.F. RecE/RecT and Redα/Redβ initiate double-stranded break repair by specifically interacting with their respective partners. Genes Dev. 14, 1971–1982 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Peset, I. & Vernos, I. The TACC proteins: TACC-ling microtubule dynamics and centrosome function. Trends Cell Biol. 18, 379–388 (2008).

    Article  CAS  Google Scholar 

  31. Giet, R. et al. Drosophila Aurora A kinase is required to localize D-TACC to centrosomes and to regulate astral microtubules. J. Cell Biol. 156, 437–451 (2002).

    Article  CAS  Google Scholar 

  32. Kinoshita, K. Aurora A phosphorylation of TACC3/maskin is required for centrosome-dependent microtubule assembly in mitosis. J. Cell Biol. 170, 1047–1055 (2005).

    Article  CAS  Google Scholar 

  33. Fu, W. et al. Clathrin recruits phosphorylated TACC3 to spindle poles for bipolar spindle assembly and chromosome alignment. J. Cell Sci. 123, 3645–3651 (2010).

    Article  CAS  Google Scholar 

  34. Royle, S.J., Bright, N.A. & Lagnado, L. Clathrin is required for the function of the mitotic spindle. Nature 434, 1152–1157 (2005).

    Article  CAS  Google Scholar 

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Acknowledgements

A.W.B. is supported by a Max Planck Society Fellowship. The research leading to these results received funding from the European Community's Seventh Framework Programmes (FP7/2007-2013) MITOSYS (Systems Biology of Mitosis) (grant number 241548), EUCOMMTOOLS (EUCOMM: Tools for Functional Annotation of the Mouse Genome) (grant number 254221) and SyBoSS (Systems Biology of Stem Cells and Reprogramming) (grant number 253422).

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  1. Marcello Maresca

    Present address: Present address: Novartis Institute for BioMedical Research, Cambridge, Massachusetts, USA.,

Authors and Affiliations

  1. Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

    Alexander W Bird & Anthony A Hyman

  2. Genomics, BioInnovationsZentrum, Technische Universität Dresden, Dresden, Germany

    Axel Erler, Jun Fu, Marcello Maresca & A Francis Stewart

  3. European Molecular Biology Laboratory, Heidelberg, Germany

    Jean-Karim Hériché

  4. Gene Bridges GmbH, Saarland University, Saarbrücken, Germany

    Youming Zhang

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Contributions

A.W.B. designed experiments, performed experiments and prepared the manuscript. A.E., J.F. and M.M. designed experiments and performed experiments. J.-K.H. developed BACFinder2.0. Y.Z. and A.A.H. designed experiments. A.F.S. designed experiments and prepared the manuscript.

Corresponding authors

Correspondence to Alexander W Bird or A Francis Stewart.

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Competing interests

Y.Z. and A.F.S. are shareholders in Gene Bridges GmbH, which holds the patent rights to the primary recombineering methodologies upon which this study is based.

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Supplementary Figures 1–3, Supplementary Table 1 and Supplementary Protocols 1 and 2. (PDF 3123 kb)

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Bird, A., Erler, A., Fu, J. et al. High-efficiency counterselection recombineering for site-directed mutagenesis in bacterial artificial chromosomes. Nat Methods 9, 103–109 (2012). https://doi.org/10.1038/nmeth.1803

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