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Controlled gene expression in primary Lgr5 organoid cultures. ​

Literature Information ​

DOI10.1038/nmeth.1802
PMID22138822
JournalNature methods
Impact Factor32.1
JCR QuartileQ1
Publication Year2011
Times Cited174
Keywordsgene expression, Lgr5 organoids, Cre recombinase
Literature TypeJournal Article, Research Support, Non-U.S. Gov't
ISSN1548-7091
Pages81-3
Issue9(1)
AuthorsBon-Kyoung Koo, Daniel E Stange, Toshiro Sato, Wouter Karthaus, Henner F Farin, Meritxell Huch, Johan H van Es, Hans Clevers

TL;DR ​

This research introduces a novel method utilizing Cre recombinase-inducible retrovirus vectors to facilitate the conditional manipulation of gene expression in primary mouse organoid cultures, addressing the challenges of traditional transgenic approaches in studying gene function in endodermal epithelia. This innovative technique offers a more efficient and less labor-intensive alternative for investigating gene roles in organs such as the stomach, small intestine, and colon.

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gene expression · Lgr5 organoids · Cre recombinase

Abstract ​

The study of gene function in endodermal epithelia such as of stomach, small intestine and colon relies heavily on transgenic approaches. Establishing such animal models is laborious, expensive and time-consuming. We present here a method based on Cre recombinase-inducible retrovirus vectors that allows the conditional manipulation of gene expression in primary mouse organoid culture systems.

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Primary Questions Addressed ​

  1. What are the advantages of using Cre recombinase-inducible retrovirus vectors over traditional transgenic approaches in gene expression studies?
  2. How does the manipulation of gene expression in organoid cultures impact the study of diseases related to endodermal epithelia?
  3. What specific challenges might researchers face when applying this method to other types of organoid cultures beyond Lgr5 organoids?
  4. In what ways can the findings from this study be translated into therapeutic strategies for gastrointestinal diseases?
  5. How does the primary mouse organoid culture system compare to other in vitro models in terms of gene expression regulation and experimental outcomes?

Key Findings ​

Key Insights:

  1. Research Background and Objective: The study addresses the challenges of investigating gene function in endodermal epithelia, specifically the stomach, small intestine, and colon. Traditional transgenic approaches to create animal models for gene manipulation are often labor-intensive, costly, and time-consuming. The objective of this research is to establish a more efficient method for conditional gene expression manipulation in primary organoid cultures derived from mouse tissue, thereby facilitating the study of gene functions in these critical biological systems.

  2. Main Methods and Findings: The researchers developed a novel technique utilizing Cre recombinase-inducible retrovirus vectors to achieve controlled gene expression in primary Lgr5-positive organoid cultures. This method allows for the targeted manipulation of specific genes within the organoids, providing a more flexible and rapid approach compared to conventional transgenic models. The study demonstrated that this system enables the conditional activation or inactivation of genes, thus allowing for the analysis of various gene functions in a controlled environment. The findings suggest that this method is effective for studying gene roles in organoid systems, which mimic the in vivo architecture and function of intestinal tissues.

  3. Core Conclusions: The study concludes that the Cre recombinase-inducible retrovirus vector system significantly enhances the ability to manipulate gene expression in primary organoid cultures. This approach allows researchers to bypass the complexities associated with traditional transgenic models, providing a robust platform for gene function studies. The conditional nature of the gene manipulation offers insights into the temporal and spatial dynamics of gene expression, thereby enriching the understanding of gene roles in the development and maintenance of endodermal epithelia.

  4. Research Significance and Impact: This research holds substantial significance as it introduces a streamlined methodology for gene manipulation in organoid cultures, which can accelerate the discovery of gene functions and their implications in gastrointestinal biology. The impact of this work extends beyond basic research; it has potential applications in understanding diseases such as cancer and inflammatory bowel disease, where gene dysregulation plays a critical role. By providing a more accessible and efficient tool for genetic studies, this method may enable researchers to uncover novel therapeutic targets and strategies for treating various gastrointestinal disorders, ultimately enhancing translational research efforts in regenerative medicine and disease modeling.

References ​

  1. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. - Toshiro Sato;Robert G Vries;Hugo J Snippert;Marc van de Wetering;Nick Barker;Daniel E Stange;Johan H van Es;Arie Abo;Pekka Kujala;Peter J Peters;Hans Clevers - Nature (2009)
  2. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. - Wim de Lau;Nick Barker;Teck Y Low;Bon-Kyoung Koo;Vivian S W Li;Hans Teunissen;Pekka Kujala;Andrea Haegebarth;Peter J Peters;Marc van de Wetering;Daniel E Stange;Johan E van Es;Daniele Guardavaccaro;Richard B M Schasfoort;Yasuaki Mohri;Katsuhiko Nishimori;Shabaz Mohammed;Albert J R Heck;Hans Clevers - Nature (2011)
  3. Growth suppression of pre-T acute lymphoblastic leukemia cells by inhibition of notch signaling. - Andrew P Weng;Yunsun Nam;Michael S Wolfe;Warren S Pear;James D Griffin;Stephen C Blacklow;Jon C Aster - Molecular and cellular biology (2003)
  4. Mouse atonal homolog 1 directs intestinal progenitors to secretory cell rather than absorptive cell fate. - Kelli L VanDussen;Linda C Samuelson - Developmental biology (2010)
  5. Improved coinfection with amphotropic pseudotyped retroviral vectors. - Yuehong Wu;David W Melton;Yong Zhang;Peter J Hornsby - Journal of biomedicine & biotechnology (2009)
  6. Notch/gamma-secretase inhibition turns proliferative cells in intestinal crypts and adenomas into goblet cells. - Johan H van Es;Marielle E van Gijn;Orbicia Riccio;Maaike van den Born;Marc Vooijs;Harry Begthel;Miranda Cozijnsen;Sylvie Robine;Doug J Winton;Freddy Radtke;Hans Clevers - Nature (2005)
  7. Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155. - Kwan-Ho Chung;Christopher C Hart;Sarmad Al-Bassam;Adam Avery;Jennifer Taylor;Paresh D Patel;Anne B Vojtek;David L Turner - Nucleic acids research (2006)
  8. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. - Toshiro Sato;Daniel E Stange;Marc Ferrante;Robert G J Vries;Johan H Van Es;Stieneke Van den Brink;Winan J Van Houdt;Apollo Pronk;Joost Van Gorp;Peter D Siersema;Hans Clevers - Gastroenterology (2011)
  9. Current issues in mouse genome engineering. - Stefan Glaser;Konstantinos Anastassiadis;A Francis Stewart - Nature genetics (2005)
  10. Identification of stem cells in small intestine and colon by marker gene Lgr5. - Nick Barker;Johan H van Es;Jeroen Kuipers;Pekka Kujala;Maaike van den Born;Miranda Cozijnsen;Andrea Haegebarth;Jeroen Korving;Harry Begthel;Peter J Peters;Hans Clevers - Nature (2007)

Literatures Citing This Work ​

  1. Fusion of intestinal epithelial cells with bone marrow derived cells is dispensable for tissue homeostasis. - Joan H de Jong;Hans M Rodermond;Cheryl D Zimberlin;Valeria Lascano;Felipe De Sousa E Melo;Dick J Richel;Jan Paul Medema;Louis Vermeulen - Scientific reports (2012)
  2. Nuclear receptor binding protein 1 regulates intestinal progenitor cell homeostasis and tumour formation. - Catherine H Wilson;Catriona Crombie;Louise van der Weyden;George Poulogiannis;Alistair G Rust;Mercedes Pardo;Tannia Gracia;Lu Yu;Jyoti Choudhary;Gino B Poulin;Rebecca E McIntyre;Douglas J Winton;H Nikki March;Mark J Arends;Andrew G Fraser;David J Adams - The EMBO journal (2012)
  3. The intestinal epithelium tuft cells: specification and function. - François Gerbe;Catherine Legraverend;Philippe Jay - Cellular and molecular life sciences : CMLS (2012)
  4. Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors. - Bon-Kyoung Koo;Maureen Spit;Ingrid Jordens;Teck Y Low;Daniel E Stange;Marc van de Wetering;Johan H van Es;Shabaz Mohammed;Albert J R Heck;Madelon M Maurice;Hans Clevers - Nature (2012)
  5. Major signaling pathways in intestinal stem cells. - Tim Vanuytsel;Stefania Senger;Alessio Fasano;Terez Shea-Donohue - Biochimica et biophysica acta (2013)
  6. Epithelial and mesenchymal contribution to the niche: a safeguard for intestinal stem cell homeostasis. - Nicholas R Smith;Paige S Davies;Alain D Silk;Melissa H Wong - Gastroenterology (2012)
  7. Restriction of intestinal stem cell expansion and the regenerative response by YAP. - Evan R Barry;Teppei Morikawa;Brian L Butler;Kriti Shrestha;Rosemarie de la Rosa;Kelley S Yan;Charles S Fuchs;Scott T Magness;Ron Smits;Shuji Ogino;Calvin J Kuo;Fernando D Camargo - Nature (2013)
  8. A gutsy task: generating intestinal tissue from human pluripotent stem cells. - Stacy R Finkbeiner;Jason R Spence - Digestive diseases and sciences (2013)
  9. Towards neuronal organoids: a method for long-term culturing of high-density hippocampal neurons. - George K Todd;Casey A Boosalis;Aaron A Burzycki;Michael Q Steinman;Lynda D Hester;Pete W Shuster;Randen L Patterson - PloS one (2013)
  10. Genetic reconstitution of tumorigenesis in primary intestinal cells. - Kunishige Onuma;Masako Ochiai;Kaoru Orihashi;Mami Takahashi;Toshio Imai;Hitoshi Nakagama;Yoshitaka Hippo - Proceedings of the National Academy of Sciences of the United States of America (2013)

... (164 more literatures)

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