Retroviral Gene Expression Control in Primary Organoid Cultures

互联网2013-12-31

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Abstract
Table of Contents
Materials
Literature Cited

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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Materials

Basic Protocol 1:

Materials

Endodermal organoids (pylorus of stomach, small intestine and colon organoids; Sato and Clevers, )
ENRWntNic medium (see recipe )
5 µM CHIR99021 (Sigma)
DMEM medium containing 10% FBS
OptiMEM (Life Technologies, cat no. 51985‐034)
1 µg/ml puromycin
10 µg/ml blasticidin
Retroviral DNA construct
1 µg/ml polyethyleneimine (PEI, Polysciences)
Infection medium (see recipe ) with and without polybrene
TrypLE (Invitrogen)
Organoid culture medium (see recipe )
Matrigel (BD Biosciences)

24‐well culture plates
150‐mm culture dishes
Refrigerated centrifuge with microtiter plate carrier
Microcentrifuge
48‐well culture plates

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Literature Cited

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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.