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Generation of transgenic mice with megabase-sized human yeast artificial chromosomes by yeast spheroplast–embryonic stem cell fusion

Nature Protocols volume 8, pages 1567–1582 (2013)Cite this article

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Abstract

Introducing human genes into mice offers the opportunity to analyze their in vivo function or to obtain therapeutic molecules. For proper gene regulation, or in case of multigene families, megabase (Mb)-sized DNA fragments often have to be used. Yeast artificial chromosome (YAC)–mediated transgenesis is irreplaceable for this purpose, because alternative methods such as the use of bacterial artificial chromosomes (BACs) cannot introduce DNA fragments larger than 500 kb into the mouse germ line. However, YAC libraries often contain only partial gene loci. Time-consuming reconstruction of YACs, genetic instability and the difficulty in obtaining intact YAC DNA above a certain size impede the generation of humanized mice. Here we describe how to reconstruct YACs containing Mb-sized human DNA, such as the T cell receptor-α (TRA) gene locus, thus facilitating the introduction of large DNA fragments into the mouse germ line. Fusion of YAC-containing yeast and embryonic stem (ES) cells avoids the need for YAC DNA purification. These ES cells are then used to stably introduce the functional TRA gene locus into the mouse germ line. The protocol takes ∼1 year to complete, from reconstruction of the entire TRA gene locus from YACs containing partial but overlapping TRA regions to germline transmission of the YAC.

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Figure 1: Identification of overlapping TRA-YACs with a set of TRA-specific primers.
Figure 2: Determination of YAC insert orientation by YAC fragmentation.
Figure 3: Schematic diagram for the transfer of one YAC to a MATα host, YAC end modification with different selectable markers and reconstruction of YACs.
Figure 4: Alu Southern blot analysis of recombined TRA-YACs.
Figure 5: Modification of an intact Mb-sized TRA-YAC.
Figure 6: Transfer of human TRA-YAC into the mouse genome by ES cell–yeast spheroplast fusion.

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Acknowledgements

We thank M. Textor for technical assistance, and G. Willimsky, T. Kammertoens, D. Strathdee and J. Guo for critical reading of the manuscript. We thank E. A. Macintyre (Hôpital Necker Enfants Malades) for YAC 27BE8 (y27BE8), isolated from the ICRF human YAC library. We also thank A. Garratt (Max Delbrük Center) for the ES14.1 cell line. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich TR36) and the 'Alliance' program of the Helmholtz-Gemeinschaft Deutscher Forschungszentren (HA-202).

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Authors and Affiliations

  1. Max Delbrück Center for Molecular Medicine, Berlin, Germany

    Liangping Li & Thomas Blankenstein

  2. Disease Model Animal Center, Institutes of Clinical and Translational Medicine, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China

    Liangping Li

  3. Institute of Immunology, Charité, Campus Berlin Buch, Berlin, Germany

    Thomas Blankenstein

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L.L. designed the protocol, performed the experiments, analyzed the data and wrote the manuscript. T.B. supervised the project, analyzed the data and wrote the manuscript.

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Correspondence to Liangping Li.

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Li, L., Blankenstein, T. Generation of transgenic mice with megabase-sized human yeast artificial chromosomes by yeast spheroplast–embryonic stem cell fusion. Nat Protoc 8, 1567–1582 (2013). https://doi.org/10.1038/nprot.2013.093

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