A protocol for lentiviral transduction and downstream analysis of intestinal organoids.
Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.
Tytgat Institute for Liver and Intestinal Research, Department of Gasteroenterology and Hepatology, Academical Medical Center.
Jooske F
JF
Van Lidth de Jeude
Tytgat Institute for Liver and Intestinal Research, Department of Gasteroenterology and Hepatology, Academical Medical Center.
Jacqueline L M
JL
Vermeulen
Tytgat Institute for Liver and Intestinal Research, Department of Gasteroenterology and Hepatology, Academical Medical Center.
Paula S
PS
Montenegro-Miranda
Tytgat Institute for Liver and Intestinal Research, Department of Gasteroenterology and Hepatology, Academical Medical Center.
Gijs R
GR
Van den Brink
Tytgat Institute for Liver and Intestinal Research, Department of Gasteroenterology and Hepatology, Academical Medical Center; j.heijmans@amc.nl.
Jarom
J
Heijmans
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D055785:Gene Knockdown Techniques / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D007413:Intestinal Mucosa / Q000166:cytology / Q000502:physiology* / Q000821:virology; D007421:Intestine, Small / Q000166:cytology / Q000502:physiology* / Q000821:virology; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D009924:Organ Culture Techniques; D009940:Organoids / Q000502:physiology / Q000821:virology; D034622:RNA Interference; D013234:Stem Cells / Q000166:cytology / Q000821:virology; D014161:Transduction, Genetic / Q000379:methods*; D019076:Transgenes