Journal
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Volume -, Issue 90, Pages -Publisher
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/51765
Keywords
Genetics; Issue 90; Retrovirus; Lentivirus; Organoid culture; Lgr5; Intestine; 3Rs
Categories
Funding
- Wellcome Trust
- Medical Research Council (MRC)
- Medical Research Council [1357931] Funding Source: researchfish
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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.
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