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Rapid bacterial artificial chromosome modification for large-scale mouse transgenesis

Nature Protocols volume 5, pages 1678–1696 (2010)Cite this article

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Abstract

We report here a high-throughput method for the modification of bacterial artificial chromosomes (BACs) that uses a novel two-plasmid approach. In this protocol, a vector modified in our laboratory to hold an R6Kγ origin of replication and a marker recombination cassette is inserted into a BAC in a single recombination step. Temporal control of recombination is achieved through the use of a second plasmid, pSV1.RecA, which possesses a recombinase gene and a temperature-sensitive origin of replication. This highly efficient protocol has allowed us to successfully modify more than 2,000 BACs, from which over 1,000 BAC transgenic mice have been generated. A complete cycle from BAC choice to embryo implantation takes about 5 weeks. Marker genes introduced into the mice include EGFP and EGFP-L10a. All vectors used in this project can be obtained from us by request, and the EGFP reporter mice are available through the Mutant Mouse Regional Resource Center (NINDS/GENSAT collection). CNS anatomical expression maps of the mice are available to the public at http://www.gensat.org/.

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Figure 1: A flowchart of the two-plasmid/one–recombination step approach to BAC modification.
Figure 2: A diagrammatic representation of the two-plasmid/one–recombination step BAC modification protocol.
Figure 3: Diagrammatic representation of an A-homology (A-box) arm.
Figure 4: Confocal micrographs of direct epifluorescence captured from tissue sections from six GENSAT EGFP BAC transgenic mice that were generated using the protocol described in this paper.
Figure 5: Ethidium bromide–stained agarose gel pattern showing digested pLD53.
Figure 6: Pulsed-field gel patterns of linearized modified BAC DNA.

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Acknowledgements

We acknowledge C. Wang and S. Mehta for their valuable contribution to the development of the protocol described in this report and we thank S. Mehta and M. Almahariq for critical reading of the paper. We thank Z.D. Barrera for assistance with Figure 2 and J. Walsh for her untiring help with plasmid distribution to the scientific community. This work was supported by an NIH/NINDS contract N01 NS-7-2370, the Simons Foundation; N.H. is a Howard Hughes Medical Institute investigator.

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Authors and Affiliations

  1. The GENSAT Project, The Rockefeller University, New York, New York, USA

    Shiaoching Gong, Laura Kus & Nathaniel Heintz

  2. Laboratory of Molecular Biology, The Rockefeller University, New York, New York, USA

    Shiaoching Gong & Nathaniel Heintz

  3. Howard Hughes Medical Institute, The Rockefeller University, New York, New York, USA

    Nathaniel Heintz

Authors
  1. Shiaoching Gong
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Contributions

N.H. conceptualized and directed the project. N.H. and S.G. developed the high-throughput method of BAC modification that uses a single recombination step. S.G. designed and conducted experiments. S.G. analyzed data; L.K. improved quality, condensed and summarized the protocol and wrote the paper; L.K. performed confocal microscopy.

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Correspondence to Shiaoching Gong or Nathaniel Heintz.

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The authors declare no competing financial interests.

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Gong, S., Kus, L. & Heintz, N. Rapid bacterial artificial chromosome modification for large-scale mouse transgenesis. Nat Protoc 5, 1678–1696 (2010). https://doi.org/10.1038/nprot.2010.131

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