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Using the GEMM-ESC strategy to study gene function in mouse models

Nature Protocols volume 10, pages 1755–1785 (2015)Cite this article

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Abstract

Preclinical in vivo validation of target genes for therapeutic intervention requires careful selection and characterization of the most suitable animal model in order to assess the role of these genes in a particular process or disease. To this end, genetically engineered mouse models (GEMMs) are typically used. However, the appropriate engineering of these models is often cumbersome and time consuming. Recently, we and others described a modular approach for fast-track modification of existing GEMMs by re-derivation of embryonic stem cells (ESCs) that can be modified by recombinase-mediated transgene insertion and subsequently used for the production of chimeric mice. This 'GEMM-ESC strategy' allows for rapid in vivo analysis of gene function in the chimeras and their offspring. Moreover, this strategy is compatible with CRISPR/Cas9-mediated genome editing. This protocol describes when and how to use the GEMM-ESC strategy effectively, and it provides a detailed procedure for re-deriving and manipulating GEMM-ESCs under feeder- and serum-free conditions. This strategy produces transgenic mice with the desired complex genotype faster than traditional methods: generation of validated GEMM-ESC clones for controlled transgene integration takes 9–12 months, and recombinase-mediated transgene integration and chimeric cohort production takes 2–3 months. The protocol requires skills in embryology, stem cell biology and molecular biology, and it is ideally performed within, or in close collaboration with, a transgenic facility.

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Figure 1: Three routes to introduce a modified allele on a GEMM background: the zygote route, the conventional ESC route and the GEMM-ESC route.
Figure 2: Time investment and the number of mice needed to obtain an experimental cohort of 12 mice containing a new transgene (Tg) on a mutant background varying from one (XF/+) to five modified alleles (XF/F;YF/F;ZF/+).
Figure 3: Schematic depiction of the GEMM-ESC protocol divided into three parts: ESC derivation—Steps 1–24; targeting of the Col1a1 locus with a docking site containing a neomycin resistance cassette flanked by Frt sites followed by a promoter-less hygromycin gene—Steps 25–54; and Flp-mediated integration of a transgene-coding shuttle vector for conditional overexpression of a cDNA and a luciferase gene, Steps 55–76.
Figure 4: Col1a1-targeting vectors and compatible Flp-in vectors.
Figure 5: Establishing ESC clones from KrasLSL-G12D/+ embryos.
Figure 6: Verification of targeting and Flp-in integration in ESC clones by Southern blot analysis.
Figure 7: Workflow and decision tree for the in vivo validation of the various genetic modifications introduced in a particular GEMM using the GEMM-ESC approach.

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Acknowledgements

This work was supported by grants from the Dutch Cancer Society (KWF), the Netherlands Organization for Scientific Research (NWO) and the European Research Council (ERC), by a National Roadmap grant for Large-Scale Research Facilities provided by NWO, by the Cancer Systems Biology Center funded by NWO and by the EuroSyStem, Infrafrontier and EurocanPlatform projects as part of the European Union's seventh framework program. We thank J. Nichols and A. Smith (Wellcome Trust Centre for Stem Cell Research) for introducing us to their ESC derivation and ESC culture procedures, which form the basis for this protocol. We thank R. Jaenisch (Whitehead Institute for Biomedical Research) for the Flp-in plasmids. We thank F. van der Ahé, M. Cozijnsen and S. Kautschitsch for their excellent technical assistance; M. Snoek for the official MGI nomenclature; the Genomics Core Facility for their assistance in CNV analysis; and the Infrafrontier consortium headed by M. Hrabé de Angelis for providing a platform to distribute GEMM-ESC clones.

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Authors and Affiliations

  1. Mouse Clinic for Cancer and Aging research (MCCA) Transgenic Core Facility, The Netherlands Cancer Institute, Amsterdam, The Netherlands

    Ivo J Huijbers, Jessica Del Bravo, Rahmen Bin Ali, Colin Pritchard & Tanya M Braumuller

  2. Division of Molecular Pathology and Cancer Genomics Centre Netherlands, The Netherlands Cancer Institute, Amsterdam, The Netherlands

    Martine H van Miltenburg, Linda Henneman, Ewa M Michalak, Anton Berns & Jos Jonkers

  3. Division of Molecular Genetics, The Netherlands Cancer Institute, Amsterdam, The Netherlands

    Anton Berns

  4. Skoltech Center for Stem Cell Research, Moscow Region, Russia

    Anton Berns

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  1. Ivo J Huijbers
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Contributions

I.J.H. developed the protocol. J.D.B., R.B.A., C.P. and E.M.M. contributed to and supported the development of the protocol. T.M.B., M.H.v.M. and L.H. expanded the protocol and generated the unpublished GEMM-ESC clones in Table 1. I.J.H., A.B. and J.J. designed the study and wrote the manuscript.

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Correspondence to Ivo J Huijbers or Jos Jonkers.

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Huijbers, I., Del Bravo, J., Bin Ali, R. et al. Using the GEMM-ESC strategy to study gene function in mouse models. Nat Protoc 10, 1755–1785 (2015). https://doi.org/10.1038/nprot.2015.114

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