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Cre-Recombinase Induces Apoptosis and Cell Death in Enterocyte Organoids
The culture of primary intestinal epithelia cells is not possible in a normal culture system. In 2009 a three-dimensional culture system of intestinal stem cells was established that shows many of the physiological features of the small intestine, such as crypt-villus structure, stem cell niche and...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9332190/ https://www.ncbi.nlm.nih.gov/pubmed/35892654 http://dx.doi.org/10.3390/antiox11081452 |
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author | Moll, Franziska Spaeth, Manuela Schröder, Katrin |
author_facet | Moll, Franziska Spaeth, Manuela Schröder, Katrin |
author_sort | Moll, Franziska |
collection | PubMed |
description | The culture of primary intestinal epithelia cells is not possible in a normal culture system. In 2009 a three-dimensional culture system of intestinal stem cells was established that shows many of the physiological features of the small intestine, such as crypt-villus structure, stem cell niche and all types of differentiated intestinal epithelial cells. These enteroids can be used to analyze biology of intestinal stem cells, gut homeostasis and the development of diseases. They also give the possibility to reduce animal numbers, as enteroids can be cryo-conserved and cultivated for many passages. To investigate the influence of genes such as NADPH oxidases on the gut homeostasis, transgenic approached are the method of choice. The generation of enteroids from knockout mice allows real-time observations of knockout effects. Often conditional knockout or overexpression strategies using inducible Cre recombinase are applied to avoid effects of adaption to the knockout. However, the Cre recombinase has many known caveats from unspecific binding and its endonuclease activity. In this study, we show that although NADPH oxidases are important for in vivo differentiation and proliferation of the intestine, their expression is drastically reduced in the organoid system. Activation of Cre recombinase by 4-hydroxy tamoxifen in freshly isolated enteroids, independently of floxed genes, leads to decreased diameter of organoids. This effect is concentration-dependent and is caused by reduced cell proliferation and induction of apoptosis and DNA damage. In contrast, constitutive expression of Cre has no impact on the enteroids. Therefore, reduction of tamoxifen concentration and treatment duration should be carefully titrated, and appropriate controls are necessary. |
format | Online Article Text |
id | pubmed-9332190 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-93321902022-07-29 Cre-Recombinase Induces Apoptosis and Cell Death in Enterocyte Organoids Moll, Franziska Spaeth, Manuela Schröder, Katrin Antioxidants (Basel) Article The culture of primary intestinal epithelia cells is not possible in a normal culture system. In 2009 a three-dimensional culture system of intestinal stem cells was established that shows many of the physiological features of the small intestine, such as crypt-villus structure, stem cell niche and all types of differentiated intestinal epithelial cells. These enteroids can be used to analyze biology of intestinal stem cells, gut homeostasis and the development of diseases. They also give the possibility to reduce animal numbers, as enteroids can be cryo-conserved and cultivated for many passages. To investigate the influence of genes such as NADPH oxidases on the gut homeostasis, transgenic approached are the method of choice. The generation of enteroids from knockout mice allows real-time observations of knockout effects. Often conditional knockout or overexpression strategies using inducible Cre recombinase are applied to avoid effects of adaption to the knockout. However, the Cre recombinase has many known caveats from unspecific binding and its endonuclease activity. In this study, we show that although NADPH oxidases are important for in vivo differentiation and proliferation of the intestine, their expression is drastically reduced in the organoid system. Activation of Cre recombinase by 4-hydroxy tamoxifen in freshly isolated enteroids, independently of floxed genes, leads to decreased diameter of organoids. This effect is concentration-dependent and is caused by reduced cell proliferation and induction of apoptosis and DNA damage. In contrast, constitutive expression of Cre has no impact on the enteroids. Therefore, reduction of tamoxifen concentration and treatment duration should be carefully titrated, and appropriate controls are necessary. MDPI 2022-07-26 /pmc/articles/PMC9332190/ /pubmed/35892654 http://dx.doi.org/10.3390/antiox11081452 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Moll, Franziska Spaeth, Manuela Schröder, Katrin Cre-Recombinase Induces Apoptosis and Cell Death in Enterocyte Organoids |
title | Cre-Recombinase Induces Apoptosis and Cell Death in Enterocyte Organoids |
title_full | Cre-Recombinase Induces Apoptosis and Cell Death in Enterocyte Organoids |
title_fullStr | Cre-Recombinase Induces Apoptosis and Cell Death in Enterocyte Organoids |
title_full_unstemmed | Cre-Recombinase Induces Apoptosis and Cell Death in Enterocyte Organoids |
title_short | Cre-Recombinase Induces Apoptosis and Cell Death in Enterocyte Organoids |
title_sort | cre-recombinase induces apoptosis and cell death in enterocyte organoids |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9332190/ https://www.ncbi.nlm.nih.gov/pubmed/35892654 http://dx.doi.org/10.3390/antiox11081452 |
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