Retroviral Gene Expression Control in Primary Organoid Cultures
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Abstract
In this unit, we describe a protocol for regulated gene expression in primary endodermal organoid culture using retroviral vectors. The study of gene function in endodermal epithelia such as those lining the stomach, small intestine, and colon has so far mainly relied on the generation of transgenic mouse lines. Establishing such animal models is laborious, expensive, and time?consuming. Ever?expanding endodermal organoids, grown in an in vitro 3?D epithelial culture system, faithfully recapitulate the in vivo counterpart and represent a sustainable alternative. Gene overexpression and knockdown can be achieved in organoids by retroviral transduction. The technique can also be applied to organoids derived from pre?established mutant mouse lines or used in combination with chemical and biological inhibitors or activators. This method provides a novel, versatile tool for phenotypic analysis of endodermal epithelium in vitro. Curr. Protoc. Stem Cell Biol . 27:5A.6.1?5A.6.8. © 2013 by John Wiley & Sons, Inc.
Keywords: organoid culture; Lgr5?positive stem cells; retrovirus; transgenic animal model; 3R
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| Sato, T., Stange, D.E., Ferrante, M., Vries, R.G., Van Es, J.H., Van den Brink, S., Van Houdt, W.J., Pronk, A., Van Gorp, J., Siersema, P.D., and Clevers, H. 2011. Long‐term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 141:1762‐1772. | |
| Sato, T., Vries, R.G., Snippert, H.J., van de Wetering, M., Barker, N., Stange, D.E., van Es, J.H., Abo, A., Kujala, P., Peters, P.J., and Clevers, H. 2009. Single Lgr5 stem cells build crypt‐villus structures in vitro without a mesenchymal niche. Nature 459:262‐265. | |
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| Internet Resources | |
| http://www.addgene.org/browse/article/4977/ | |
| Retroviral vectors for Cre‐inducible gene overexpression and knockdown have been deposited in Addgene. |
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