Using a Novel Gut Culture System to Analyze the Influence of Known and Novel Genes on Intestinal Epithelial Differentiation

We aim to study the development of endoderm and the differentiation of the intestinal epithelium in mice, using embryonic gut culture, ES cell differentiation, and mouse models. We have established an innovative mouse embryonic gut culture system (caternary culture) in our laboratory that maintains the three-dimensional architecture of the gut. This provides an opportunity to directly study the function of genes in a system that closely mimics the development and differentiation of the gut in vivo. The development of the epithelial cell layer in caternary cultures has been studied in detail using light and electron microscopy and immunohistochemistry for molecular markers of gut development. We have also demonstrated that plasmids encoding genes can be introduced into the epithelial cell layer using a low-voltage electroporation technique. The effects of expressing mutant versions of components of the APC, Ras, and TGF[Beta] signaling pathways on intestinal cell biology will be examined using this system. Along side this work, we also aim to identify novel genes that can promote intestinal differentiation using a unique screening strategy in embryonic stem (ES) cells. This is based on a unique ES cell line generated in our laboratory that contains a LacZ marker gene under the control of the A33 promoter (a definitive marker of intestinal epithelium). The ultimate objective of this project is to screen an expression library for genes that can promote intestinal differentiation.