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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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.
The authors declare no competing financial interests.
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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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