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

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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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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Authors

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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