Retroviral Gene Expression Control in Primary Organoid Cultures

Bon‐Kyoung Koo1, Valentina Sasselli1, Hans Clevers1

1 Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences, and University Medical Center Utrecht, Utrecht, The Netherlands
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 5A.6
DOI:  10.1002/9780470151808.sc05a06s27
Online Posting Date:  November, 2013
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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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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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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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Figures

Videos

Literature Cited

  Barker, N., van Es, J.H., Kuipers, J., Kujala, P., van den Born, M., Cozijnsen, M., Haegebarth, A., Korving, J., Begthel, H., Peters, P.J., and Clevers, H. 2007. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449:1003‐1007.
  Barker, N., Huch, M., Kujala, P., van de Wetering, M., Snippert, H.J., van Es, J.H., Sato, T., Stange, D.E., Begthel, H., van den Born, M., Danenberg, E., van den Brink, S., Korving, J., Abo, A., Peters, P.J., Wright, N., Poulsom, R., and Clevers, H. 2010. Lgr5(+ve) stem cells drive self‐renewal in the stomach and build long‐lived gastric units in vitro. Cell Stem Cell 6:25‐36.
  de Lau, W., Barker, N., Low, T.Y., Koo, B.K., Li, V.S., Teunissen, H., Kujala, P., Haegebarth, A., Peters, P.J., van de Wetering, M., Stange, D.E., van Es, J.E., Guardavaccaro, D., Schasfoort, R.B., Mohri, Y., Nishimori, K., Mohammed, S., Heck, A.J., and Clevers, H. 2011. Lgr5 homologues associate with Wnt receptors and mediate R‐spondin signalling. Nature 476: 293‐297.
  Huch, M., Dorrell, C., Boj, S.F., van Es, J.H., Li, V.S., van de Wetering, M., Sato, T., Hamer, K., Sasaki, N., Finegold, M.J., Haft, A., Vries, R.G., Grompe, M., and Clevers, H. 2013. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt‐driven regeneration. Nature 494:247‐250.
  Koo, B.K., Stange, D.E., Sato, T., Karthaus, W., Farin, H.F., Huch, M., van Es, J.H., and Clevers, H. 2011. Controlled gene expression in primary Lgr5 organoid cultures. Nat. Methods 9:81‐83.
  Koo, B.K., Spit, M., Jordens, I., Low, T.Y., Stange, D.E., van de Wetering, M., van Es, J.H., Mohammed, S., Heck, A.J., Maurice, M.M., and Clevers, H. 2012. Tumour suppressor RNF43 is a stem‐cell E3 ligase that induces endocytosis of Wnt receptors. Nature 488:665‐669.
  Morita, S., Kojima, T., and Kitamura, T. 2000. Plat‐E: An efficient and stable system for transient packaging of retroviruses. Gene Ther. 7:1063‐1066.
  Mustata, R.C., Van Loy, T., Lefort, A., Libert, F., Strollo, S., Vassart, G., and Garcia, M.I. 2011. Lgr4 is required for Paneth cell differentiation and maintenance of intestinal stem cells ex vivo. EMBO Rep. 12:558‐564.
  Sato, T. and Clevers, H. 2013a. Growing self‐organizing mini‐guts from a single intestinal stem cell: Mechanism and applications. Science 340:1190‐1194.
  Sato, T. and Clevers, H. 2013b. Primary mouse small intestinal epithelial cell cultures. Methods Mol. Biol. 945:319‐328.
  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.
  Wu, Y., Melton, D.W., Zhang, Y., and Hornsby, P.J. 2009. Improved coinfection with amphotropic pseudotyped retroviral vectors. J. Biomed. Biotechnol. 2009:901079.
  Yui, S., Nakamura, T., Sato, T., Nemoto, Y., Mizutani, T., Zheng, X., Ichinose, S., Nagaishi, T., Okamoto, R., Tsuchiya, K., Clevers, H., and Watanabe, M. 2012. Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5(+) stem cell. Nat. Med. 18:618‐623.
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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