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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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References

  1. Sato, T. & Clevers, H. Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science 340, 1190–1194 (2013).

    Article  CAS  PubMed  Google Scholar 

  2. Vidi, P.A., Bissell, M.J. & Lelievre, S.A. Three-dimensional culture of human breast epithelial cells: the how and the why. Methods Mol. Biol. 945, 193–219 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. Sanchez Alvarado, A. & Yamanaka, S. Rethinking differentiation: stem cells, regeneration, and plasticity. Cell 157, 110–119 (2014).

    Article  CAS  PubMed  Google Scholar 

  4. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

    Article  CAS  PubMed  Google Scholar 

  5. Frese, K.K. & Tuveson, D.A. Maximizing mouse cancer models. Nat. Rev. Cancer 7, 645–658 (2007).

    Article  CAS  PubMed  Google Scholar 

  6. Dranoff, G. Experimental mouse tumour models: what can be learnt about human cancer immunology? Nat. Rev. Immunol. 12, 61–66 (2012).

    Article  CAS  Google Scholar 

  7. Couzin-Frankel, J. Breakthrough of the year 2013. Cancer immunotherapy. Science 342, 1432–1433 (2013).

    Article  CAS  PubMed  Google Scholar 

  8. Camacho, L.H. CTLA-4 blockade with ipilimumab: biology, safety, efficacy, and future considerations. Cancer Med. 4, 661–672 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Huijbers, I.J., Krimpenfort, P., Berns, A. & Jonkers, J. Rapid validation of cancer genes in chimeras derived from established genetically engineered mouse models. Bioessays 33, 701–710 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Henneman, L. et al. Selective resistance to the PARP inhibitor olaparib in a mouse model for BRCA1-deficient metaplastic breast cancer. Proc. Natl. Acad. Sci. USA 112, 8409–8414 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Huijbers, I.J. et al. Rapid target gene validation in complex cancer mouse models using re-derived embryonic stem cells. EMBO Mol. Med. 6, 212–225 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Ivics, Z. et al. Germline transgenesis in rabbits by pronuclear microinjection of Sleeping Beauty transposons. Nat. Protoc. 9, 794–809 (2014).

    Article  CAS  PubMed  Google Scholar 

  13. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Miller, J.C. et al. A TALE nuclease architecture for efficient genome editing. Nat. Biotechnol. 29, 143–148 (2011).

    Article  CAS  PubMed  Google Scholar 

  16. Ran, F.A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Urnov, F.D., Rebar, E.J., Holmes, M.C., Zhang, H.S. & Gregory, P.D. Genome editing with engineered zinc finger nucleases. Nat. Rev. Genet. 11, 636–646 (2010).

    Article  CAS  PubMed  Google Scholar 

  18. Zhang, F. et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat. Biotechnol. 29, 149–153 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Yang, H. et al. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154, 1370–1379 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Silva, J. et al. Promotion of reprogramming to ground state pluripotency by signal inhibition. PLoS Biol. 6, e253 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Ying, Q.L. et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Czechanski, A. et al. Derivation and characterization of mouse embryonic stem cells from permissive and nonpermissive strains. Nat. Protoc. 9, 559–574 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Hanna, J. et al. Metastable pluripotent states in NOD-mouse-derived ESCs. Cell Stem Cell 4, 513–524 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Nichols, J. et al. Validated germline-competent embryonic stem cell lines from nonobese diabetic mice. Nat. Med. 15, 814–818 (2009).

    Article  CAS  PubMed  Google Scholar 

  26. Reinholdt, L.G. et al. Generating embryonic stem cells from the inbred mouse strain DBA/2J, a model of glaucoma and other complex diseases. PLoS ONE 7, e50081 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Premsrirut, P.K. et al. A rapid and scalable system for studying gene function in mice using conditional RNA interference. Cell 145, 145–158 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Saborowski, M. et al. A modular and flexible ESC-based mouse model of pancreatic cancer. Genes Dev. 28, 85–97 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. van Miltenburg, M.H. & Jonkers, J. Using genetically engineered mouse models to validate candidate cancer genes and test new therapeutic approaches. Curr. Opin. Genet. Dev. 22, 21–27 (2012).

    Article  CAS  PubMed  Google Scholar 

  30. Beard, C., Hochedlinger, K., Plath, K., Wutz, A. & Jaenisch, R. Efficient method to generate single-copy transgenic mice by site-specific integration in embryonic stem cells. Genesis 44, 23–28 (2006).

    Article  CAS  PubMed  Google Scholar 

  31. Premsrirut, P.K., Dow, L.E., Park, Y., Hannon, G.J. & Lowe, S.W. Creating transgenic shRNA mice by recombinase-mediated cassette exchange. Cold Spring Harb. Protoc. 2013, 835–842 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Hsu, P.D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Pattanayak, V. et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat. Biotechnol. 31, 839–843 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Smith, C. et al. Whole-genome sequencing analysis reveals high specificity of CRISPR/Cas9 and TALEN-based genome editing in human iPSCs. Cell Stem Cell 15, 12–13 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Veres, A. et al. Low incidence of off-target mutations in individual CRISPR-Cas9 and TALEN targeted human stem cell clones detected by whole-genome sequencing. Cell Stem Cell 15, 27–30 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Ran, F.A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Lloyd, K., Franklin, C., Lutz, C. & Magnuson, T. Reproducibility: use mouse biobanks or lose them. Nature 522, 151–153 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Dow, L.E. et al. Conditional reverse tet-transactivator mouse strains for the efficient induction of TRE-regulated transgenes in mice. PLoS ONE 9, e95236 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Dow, L.E. et al. A pipeline for the generation of shRNA transgenic mice. Nat. Protoc. 7, 374–393 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kiyonari, H., Kaneko, M., Abe, S. & Aizawa, S. Three inhibitors of FGF receptor, ERK, and GSK3 establishes germline-competent embryonic stem cells of C57BL/6N mouse strain with high efficiency and stability. Genesis 48, 317–327 (2010).

    CAS  PubMed  Google Scholar 

  43. Tamm, C., Pijuan Galito, S. & Anneren, C. A comparative study of protocols for mouse embryonic stem cell culturing. PLoS ONE 8, e81156 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. Liang, Q., Conte, N., Skarnes, W.C. & Bradley, A. Extensive genomic copy number variation in embryonic stem cells. Proc. Natl. Acad. Sci. USA 105, 17453–17456 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Pettitt, S.J. et al. Agouti C57BL/6N embryonic stem cells for mouse genetic resources. Nat. Methods 6, 493–495 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Bin Ali, R. et al. Improved pregnancy and birth rates with routine application of nonsurgical embryo transfer. Transgenic Res. 23, 691–695 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. te Riele, H., Maandag, E.R. & Berns, A. Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs. Proc. Natl. Acad. Sci. USA 89, 5128–5132 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Heiman, M. et al. A translational profiling approach for the molecular characterization of CNS cell types. Cell 135, 738–748 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Krimpenfort, P. et al. p15Ink4b is a critical tumour suppressor in the absence of p16Ink4a. Nature 448, 943–946 (2007).

    Article  CAS  PubMed  Google Scholar 

  50. Solter, D. & Knowles, B.B. Immunosurgery of mouse blastocyst. Proc. Natl. Acad. Sci. USA 72, 5099–5102 (1975).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. McFarlane, L., Truong, V., Palmer, J.S. & Wilhelm, D. Novel PCR assay for determining the genetic sex of mice. Sex. Dev. 7, 207–211 (2013).

    Article  CAS  PubMed  Google Scholar 

  52. Hogan, B., Beddington, R., Costantini, F. & Lacy, E. Manipulating the mouse embryo. 2nd edn. (Cold Spring Harbor Laboratory Press, 1994).

  53. Wong, K. et al. Sequencing and characterization of the FVB/NJ mouse genome. Genome Biol. 13, R72 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  54. Yalcin, B., Adams, D.J., Flint, J. & Keane, T.M. Next-generation sequencing of experimental mouse strains. Mamm. Genome 23, 490–498 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Yalcin, B. et al. The fine-scale architecture of structural variants in 17 mouse genomes. Genome Biol. 13, R18 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Meuwissen, R. et al. Induction of small cell lung cancer by somatic inactivation of both Trp53 and Rb1 in a conditional mouse model. Cancer Cell 4, 181–189 (2003).

    Article  CAS  PubMed  Google Scholar 

  57. Jackson, E.L. et al. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev. 15, 3243–3248 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Jongsma, J. et al. A conditional mouse model for malignant mesothelioma. Cancer Cell 13, 261–271 (2008).

    Article  CAS  PubMed  Google Scholar 

  59. Liu, X. et al. Somatic loss of BRCA1 and p53 in mice induces mammary tumors with features of human BRCA1-mutated basal-like breast cancer. Proc. Natl. Acad. Sci. USA 104, 12111–12116 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Derksen, P.W. et al. Mammary-specific inactivation of E-cadherin and p53 impairs functional gland development and leads to pleomorphic invasive lobular carcinoma in mice. Dis. Model Mech. 4, 347–358 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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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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