title: The Generation of Organoids for Studying Wnt Signaling.
abstract: We established an in vitro culture model in which intestinal epithelial stem cells can grow into three-dimensional, ever-expanding epithelial organoids that retain their original organ identity and genetic stability. Moreover, organoids can easily be genetically modified using different genome modification strategies, including viral delivery of transgenes and CRISPR/Cas9 technology. These combined characteristics make them a useful in vitro model system to study many biological processes including the contribution of cellular signaling pathways to tissue homeostasis and disease. Here we describe our current laboratory protocols to establish human intestinal organoids and how to genetically modify both mouse and human intestinal organoids to study cellular signaling pathways, specifically Wnt signaling. Moreover, we provide a detailed protocol for lentiviral transduction and CRISPR/Cas9-mediated genome modification of organoid cultures.
authors: Hubrecht Institute-KNAW and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands.
authors: Jarno
authors: J
authors: Drost
authors: Hubrecht Institute-KNAW and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands.
authors: Benedetta
authors: B
authors: Artegiani
authors: Hubrecht Institute-KNAW and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands. h.clevers@hubrecht.eu.
authors: Hans
authors: H
authors: Clevers
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D009940:Organoids / Q000166:cytology* / Q000378:metabolism; D060449:Wnt Signaling Pathway / Q000235:genetics*
keywords: CRISPR/Cas9; Intestine; Lentivirus; Organoids; Wnt signaling