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 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.


CONCLUSIONS
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.
Quinlan
Jonathan M
JM
Centre for Regenerative Medicine, Department of Biology & Biochemistry, University of Bath, Bath BA2 7AY, UK.
Yu
Wei-Yuan
WY
Hornsey
Mark A
MA
Tosh
David
D
Slack
Jonathan M W
JM
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002478:Cells, Cultured; D018274:Electroporation; D017930:Genes, Reporter; D005822:Genetic Vectors; D007413:Intestinal Mucosa / Q000166:cytology* / Q000196:embryology* / Q000378:metabolism; D051379:Mice; D014162:Transfection
D015415:Biomarkers