Skip to main content

Generation and Analysis of Mouse Intestinal Tumors and Organoids Harboring APC and K-Ras Mutations

  • Protocol
  • First Online:
Mouse Models of Cancer

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1267))

Abstract

Genetically engineered mouse models of intestinal cancer are experimental systems in which mice are genetically manipulated to develop malignancies in the gastrointestinal tract. These models enable researchers to study the mechanisms of onset, progression, and metastasis of the disease. They also provide a valuable biological system which is suitable for testing (novel) drugs in vivo. Recently, an in vitro culture model has been established in which intestinal epithelial stem cells can grow into three-dimensional, ever-expanding epithelial organoids that retain their original organ identity and genetic stability. This culture system has been applied to diseased epithelia, such as adenoma, adenocarcinoma, and Barrett’s epithelium. These organoids can be particularly useful for studying the mechanisms of intestinal tumors and to test (novel) drugs in vitro. Here, we describe our current laboratory protocols to generate and analyze intestinal tumors and organoids harboring APC and K-Ras double mutations.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Noah TK, Donahue B, Shroyer NF (2011) Intestinal development and differentiation. Exp Cell Res 317(19):2702–2710. doi:10.1016/j.yexcr.2011.09.006

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  2. Clevers H (2013) The intestinal crypt, a prototype stem cell compartment. Cell 154(2):274–284. doi:10.1016/j.cell.2013.07.004

    Article  CAS  PubMed  Google Scholar 

  3. Clevers H, Nusse R (2012) Wnt/beta-catenin signaling and disease. Cell 149(6):1192–1205. doi:10.1016/j.cell.2012.05.012

    Article  CAS  PubMed  Google Scholar 

  4. Holland JD, Klaus A, Garratt AN, Birchmeier W (2013) Wnt signaling in stem and cancer stem cells. Curr Opin Cell Biol 25(2):254–264. doi:10.1016/j.ceb.2013.01.004

    Article  CAS  PubMed  Google Scholar 

  5. Poulogiannis G, Luo FJ, Arends MJ (2012) RAS signalling in the colorectum in health and disease. Cell Commun Adhes 19(1):1–9. doi:10.3109/15419061.2011.649380

    Article  CAS  PubMed  Google Scholar 

  6. Velho S, Haigis KM (2011) Regulation of homeostasis and oncogenesis in the intestinal epithelium by Ras. Exp Cell Res 317(19):2732–2739. doi:10.1016/j.yexcr.2011.06.002

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. van de Wetering M, Sancho E, Verweij C, de Lau W, Oving I, Hurlstone A, van der Horn K, Batlle E, Coudreuse D, Haramis AP, Tion-Pon-Fong M, Moerer P, van den Born M, Soete G, Pals S, Eilers M, Medema R, Clevers H (2002) The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell 111(2):241–250. doi:10.1016/S0092-8674(02)01014-0

    Article  PubMed  Google Scholar 

  8. Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M, Haegebarth A, Korving J, Begthel H, Peters PJ, Clevers H (2007) Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449(7165):1003–1007. doi:10.1038/Nature06196

    Article  CAS  PubMed  Google Scholar 

  9. Barker N, Ridgway RA, van Es JH, van de Wetering M, Begthel H, van den Born M, Danenberg E, Clarke AR, Sansom OJ, Clevers H (2009) Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 457(7229):608–611. doi:10.1038/Nature07602

    Article  CAS  PubMed  Google Scholar 

  10. Bouabe H, Okkenhaug K (2013) Gene targeting in mice: a review. Methods Mol Biol 1064:315–336. doi:10.1007/978-1-62703-601-6_23

    Article  PubMed  Google Scholar 

  11. Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, van Es JH, Abo A, Kujala P, Peters PJ, Clevers H (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459(7244):262–265. doi:10.1038/Nature07935

    Article  CAS  PubMed  Google Scholar 

  12. Sato T, Clevers H (2013) Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science 340(6137):1190–1194. doi:10.1126/science.1234852

    Article  CAS  PubMed  Google Scholar 

  13. Sato T, Stange DE, Ferrante M, Vries RGJ, van Es JH, van den Brink S, van Houdt WJ, Pronk A, van Gorp J, Siersema PD, Clevers H (2011) Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141(5):1762–1772. doi:10.1053/j.gastro.2011.07.050

    Article  CAS  PubMed  Google Scholar 

  14. Koo BK, Stange DE, Sato T, Karthaus W, Farin HF, Huch M, van Es JH, Clevers H (2012) Controlled gene expression in primary Lgr5 organoid cultures. Nat Methods 9(1):81–83. doi:10.1038/Nmeth.1802

    Article  CAS  Google Scholar 

  15. Schwank G, Andersson-Rolf A, Koo BK, Sasaki N, Clevers H (2013) Generation of BAC transgenic epithelial organoids. PLoS One 8(10):e76871. doi:10.1371/journal.pone.0076871

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Schwank G, Koo BK, Sasselli V, Dekkers JF, Heo I, Demircan T, Sasaki N, Boymans S, Cuppen E, van der Ent CK, Nieuwenhuis EES, Beekman JM, Clevers H (2013) Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell 13(6):653–658. doi:10.1016/j.stem.2013.11.002

    Article  CAS  PubMed  Google Scholar 

  17. Ootani A, Li XN, Sangiorgi E, Ho QT, Ueno H, Toda S, Sugihara H, Fujimoto K, Weissman IL, Capecchi MR, Kuo CJ (2009) Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nat Med 15(6):701–706. doi:10.1038/Nm.1951

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Gregorieff A, Clevers H (2010) In situ hybridization to identify gut stem cells. Curr Protoc Stem Cell Biol Chapter 2:Unit 2F 1. doi:10.1002/9780470151808.sc02f01s12

    Google Scholar 

Download references

Acknowledgements

The authors like to thank Harry Begthel, Marc van de Wetering, Stieneke van den Brink, and Jeroen Korving for their help in preparing this book chapter.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Hans Clevers .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

van Es, J.H., Clevers, H. (2015). Generation and Analysis of Mouse Intestinal Tumors and Organoids Harboring APC and K-Ras Mutations. In: Eferl, R., Casanova, E. (eds) Mouse Models of Cancer. Methods in Molecular Biology, vol 1267. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2297-0_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2297-0_6

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2296-3

  • Online ISBN: 978-1-4939-2297-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics