Generation and Analysis of Mouse Intestinal Tumors and Organoids Harboring APC and K-Ras Mutations
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.
Key wordsIntestine Lgr5 Stem-cells Cancer APC K-Ras Organoids
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.
- 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 PubMedCrossRefGoogle 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 PubMedCrossRefGoogle 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 PubMedCrossRefGoogle 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.sc02f01s12Google Scholar