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In vitro culture of embryonic mouse intestinal epithelium: cell differentiation and introduction of reporter genes

BACKGROUND: Study of the normal development of the intestinal epithelium has been hampered by a lack of suitable model systems, in particular ones that enable the introduction of exogenous genes. Production of such a system would advance our understanding of normal epithelial development and help to...

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Autores principales: Quinlan, Jonathan M, Yu, Wei-Yuan, Hornsey, Mark A, Tosh, David, Slack, Jonathan MW
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2006
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1489925/
https://www.ncbi.nlm.nih.gov/pubmed/16725020
http://dx.doi.org/10.1186/1471-213X-6-24
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author Quinlan, Jonathan M
Yu, Wei-Yuan
Hornsey, Mark A
Tosh, David
Slack, Jonathan MW
author_facet Quinlan, Jonathan M
Yu, Wei-Yuan
Hornsey, Mark A
Tosh, David
Slack, Jonathan MW
author_sort Quinlan, Jonathan M
collection PubMed
description BACKGROUND: Study of the normal development of the intestinal epithelium has been hampered by a lack of suitable model systems, in particular ones that enable the introduction of exogenous genes. Production of such a system would advance our understanding of normal epithelial development and help to shed light on the pathogenesis of intestinal neoplasia. The criteria for a reliable culture system include the ability to perform real time observations and manipulations in vitro, the preparation of wholemounts for immunostaining and the potential for introducing genes. RESULTS: The new culture system involves growing mouse embryo intestinal explants on fibronectin-coated coverslips in basal Eagle's medium+20% fetal bovine serum. Initially the cultures maintain expression of the intestinal transcription factor Cdx2 together with columnar epithelial (cytokeratin 8) and mesenchymal (smooth muscle actin) markers. Over a few days of culture, differentiation markers appear characteristic of absorptive epithelium (sucrase-isomaltase), goblet cells (Periodic Acid Schiff positive), enteroendocrine cells (chromogranin A) and Paneth cells (lysozyme). Three different approaches were tested to express genes in the developing cultures: transfection, electroporation and adenoviral infection. All could introduce genes into the mesenchyme, but only to a small extent into the epithelium. However the efficiency of adenovirus infection can be greatly improved by a limited enzyme digestion, which makes accessible the lateral faces of cells bearing the Coxsackie and Adenovirus Receptor. This enables reliable delivery of genes into epithelial cells. CONCLUSION: We describe a new in vitro culture system for the small intestine of the mouse embryo that recapitulates its normal development. The system both provides a model for studying normal development of the intestinal epithelium and also allows for the manipulation of gene expression. The explants can be cultured for up to two weeks, they form the full repertoire of intestinal epithelial cell types (enterocytes, goblet cells, Paneth cells and enteroendocrine cells) and the method for gene introduction into the epithelium is efficient and reliable.
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spelling pubmed-14899252006-07-08 In vitro culture of embryonic mouse intestinal epithelium: cell differentiation and introduction of reporter genes Quinlan, Jonathan M Yu, Wei-Yuan Hornsey, Mark A Tosh, David Slack, Jonathan MW BMC Dev Biol Methodology Article BACKGROUND: Study of the normal development of the intestinal epithelium has been hampered by a lack of suitable model systems, in particular ones that enable the introduction of exogenous genes. Production of such a system would advance our understanding of normal epithelial development and help to shed light on the pathogenesis of intestinal neoplasia. The criteria for a reliable culture system include the ability to perform real time observations and manipulations in vitro, the preparation of wholemounts for immunostaining and the potential for introducing genes. RESULTS: The new culture system involves growing mouse embryo intestinal explants on fibronectin-coated coverslips in basal Eagle's medium+20% fetal bovine serum. Initially the cultures maintain expression of the intestinal transcription factor Cdx2 together with columnar epithelial (cytokeratin 8) and mesenchymal (smooth muscle actin) markers. Over a few days of culture, differentiation markers appear characteristic of absorptive epithelium (sucrase-isomaltase), goblet cells (Periodic Acid Schiff positive), enteroendocrine cells (chromogranin A) and Paneth cells (lysozyme). Three different approaches were tested to express genes in the developing cultures: transfection, electroporation and adenoviral infection. All could introduce genes into the mesenchyme, but only to a small extent into the epithelium. However the efficiency of adenovirus infection can be greatly improved by a limited enzyme digestion, which makes accessible the lateral faces of cells bearing the Coxsackie and Adenovirus Receptor. This enables reliable delivery of genes into epithelial cells. CONCLUSION: We describe a new in vitro culture system for the small intestine of the mouse embryo that recapitulates its normal development. The system both provides a model for studying normal development of the intestinal epithelium and also allows for the manipulation of gene expression. The explants can be cultured for up to two weeks, they form the full repertoire of intestinal epithelial cell types (enterocytes, goblet cells, Paneth cells and enteroendocrine cells) and the method for gene introduction into the epithelium is efficient and reliable. BioMed Central 2006-05-25 /pmc/articles/PMC1489925/ /pubmed/16725020 http://dx.doi.org/10.1186/1471-213X-6-24 Text en Copyright © 2006 Quinlan et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methodology Article
Quinlan, Jonathan M
Yu, Wei-Yuan
Hornsey, Mark A
Tosh, David
Slack, Jonathan MW
In vitro culture of embryonic mouse intestinal epithelium: cell differentiation and introduction of reporter genes
title In vitro culture of embryonic mouse intestinal epithelium: cell differentiation and introduction of reporter genes
title_full In vitro culture of embryonic mouse intestinal epithelium: cell differentiation and introduction of reporter genes
title_fullStr In vitro culture of embryonic mouse intestinal epithelium: cell differentiation and introduction of reporter genes
title_full_unstemmed In vitro culture of embryonic mouse intestinal epithelium: cell differentiation and introduction of reporter genes
title_short In vitro culture of embryonic mouse intestinal epithelium: cell differentiation and introduction of reporter genes
title_sort in vitro culture of embryonic mouse intestinal epithelium: cell differentiation and introduction of reporter genes
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1489925/
https://www.ncbi.nlm.nih.gov/pubmed/16725020
http://dx.doi.org/10.1186/1471-213X-6-24
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