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A protocol for constructing gene targeting vectors: generating knockout mice for the cadherin family and beyond

Nature Protocols volume 3, pages 1056–1076 (2008)Cite this article

Abstract

We describe here a streamlined procedure for targeting vector construction, which often is a limiting factor for gene targeting (knockout) technology. This procedure combines various highly efficient recombination-based cloning methods in bacteria, consisting of three steps. First step is the use of Red-pathway-mediated recombination (recombineering) to capture a genomic fragment into a Gateway-compatible vector. Second, the vector is modified by recombineering to include a positive selection gene neo, from a variety of modular reagents. Finally, through a simple in vitro Gateway recombination, the modified genomic fragment is switched into a vector that contains negative selection cassettes, as well as unique sites for linearization. To demonstrate the usefulness of this protocol, we report targeted disruptions of members of the cadherin gene family, focusing on those that have not been previously studied at the molecular genetic level. This protocol needs ∼2 weeks to construct a targeting vector, and several vectors can be easily handled simultaneously using common laboratory setup.

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Figure 1: Flow chart showing step by step how to design and construct a targeting vector: considerations before and after targeting vector cloning.
Figure 2: Two basic targeting vector designs and construction of a simple loss-of-function targeting vector.
Figure 3: Gateway-compatible vectors.
Figure 4: Schematic for the construction of conditional targeting vectors.
Figure 5: Three examples of the knockout mice generated: Fat2EGFP, Fat3nlacZ and Fat4EGFP knockout mice.
Figure 6: Expression patterns of Fat2 and Fat3 protocadherin genes revealed by knockin reporter alleles.
Figure 7: Pcdha type A and B expression patterns in the adult mouse brain revealed by staining of enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP) reporters.
Figure 8: Alkaline phosphatase (AP) as a reporter to probe gene expression patterns.

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Acknowledgements

We thank Drs. A. Francis Stewart, Neal G. Copeland and Barry L. Wanner for providing their phage recombination systems, Dr. Jan-Fang Cheng (Lawrence Berkeley National Laboratory) for providing BAC clones for the clustered Pcdh locus, Drs. Changjiang Zou and Can Li (the Wu laboratory) for help with DNA blot experiments, Haiyan Peng (the Wu laboratory) for help on targeting vector construction and members of the Capecchi laboratory for comments on the article. We are grateful for the excellent technical support from ES cell culture and mouse surgery and husbandry staff in M.R.C. lab, in particular Sheila Barnett, Lois Byers, Carol Lenz, Arunth Lingam, Karl Lustig, Julie Tomlin and Joan Shuhua. G.Y. and Q.W. are supported by an American Cancer Society grant.

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Authors and Affiliations

  1. Howard Hughes Medical Institute, University of Utah, Salt Lake City, 84112, Utah, USA

    Sen Wu & Mario R Capecchi

  2. Department of Human Genetics, University of Utah, Salt Lake City, 84112, Utah, USA

    Guoxin Ying, Qiang Wu & Mario R Capecchi

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  1. Sen Wu
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  2. Guoxin Ying
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  3. Qiang Wu
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  4. Mario R Capecchi
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Correspondence to Mario R Capecchi.

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Supplementary Table 1

Oligonucleotides used in this study (PDF 176 kb)

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Wu, S., Ying, G., Wu, Q. et al. A protocol for constructing gene targeting vectors: generating knockout mice for the cadherin family and beyond. Nat Protoc 3, 1056–1076 (2008). https://doi.org/10.1038/nprot.2008.70

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