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 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.
Metadaten
Author:Franziska MollORCiDGND, Manuela Spaeth, Katrin SchröderORCiDGND
URN:urn:nbn:de:hebis:30:3-693033
DOI:https://doi.org/10.3390/antiox11081452
ISSN:2076-3921
Parent Title (English):Antioxidants
Publisher:MDPI
Place of publication:Basel
Document Type:Article
Language:English
Date of Publication (online):2022/07/26
Date of first Publication:2022/07/26
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2024/08/19
Tag:Cre-recombinase; apoptosis; cell death; enterocytes; genomic instability; organoids
Volume:11
Issue:8, art.1452
Article Number:1452
Page Number:13
First Page:1
Last Page:13
Note:
Gefördert durch den Open-Access-Publikationsfonds der Goethe-Universität
HeBIS-PPN:521201047
Institutes:Medizin
Dewey Decimal Classification:6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit
Sammlungen:Universitätspublikationen
Open-Access-Publikationsfonds:Medizin
Licence (German):License LogoCreative Commons - CC BY - Namensnennung 4.0 International