title: A video protocol of retroviral infection in primary intestinal organoid culture.
abstract: Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these 'mini-guts', also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.
authors.affiliation: Department of Genetics, University of Cambridge; Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge.
authors.forename: Amanda
authors.initials: A
authors.lastname: Andersson-Rolf
authors.affiliation: Department of Genetics, University of Cambridge; Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge.
authors.forename: Juergen
authors.initials: J
authors.lastname: Fink
authors.affiliation: Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge.
authors.forename: Roxana C
authors.initials: RC
authors.lastname: Mustata
authors.affiliation: Department of Genetics, University of Cambridge; Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge; bkk25@cam.ac.uk.
authors.forename: Bon-Kyoung
authors.initials: BK
authors.lastname: Koo
mesh_terms: D000818:Animals; D055785:Gene Knockdown Techniques / Q000379:methods; D006801:Humans; D007421:Intestine, Small / Q000502:physiology* / Q000821:virology*; D051379:Mice; D009924:Organ Culture Techniques / Q000379:methods*; D043562:Receptors, G-Protein-Coupled; D012190:Retroviridae / Q000235:genetics*; D012192:Retroviridae Infections / Q000821:virology*; D014161:Transduction, Genetic; D019076:Transgenes
chemical_list: C116884:LGR5 protein, human; D043562:Receptors, G-Protein-Coupled