BACKGROUND AIMS: Gene-targeted and transgenic mice have provided profound insights into the genetic control of intestinal development and differentiation. Here we describe a complementary approach using an innovative embryonic gut culture system in which the tubular architecture of the gut is maintained and rapid analysis of gene function can be achieved by introduction of expression constructs into the epithelial cell layer by using low-voltage square-wave electroporation. METHODS: Electroporation of embryonic gut explants was optimized by using an electrosquareporator to vary the voltage and number of electric pulses. The pulses were applied unidirectionally to confine electroporation of plasmids to one side of the epithelium, providing experimental and control regions within the same gut segment. The expression of constructs was examined by fluorescence and confocal microscopy, immunohistochemistry, and beta-galactosidase activity assays. RESULTS: Conditions were identified in which a high proportion of cells (5 to 20 ) within the epithelial cell layer of gut explants expressed transfected genes without cell death. Electroporation of a construct encoding a Cre-enhanced green fluorescent protein (EGFP) fusion protein into explants from ROSA26 reporter animals resulted in extensive activation of beta-galactosidase expression, indicating that the electroporated construct produced functionally active protein. CONCLUSIONS: A novel embryonic gut culture system has been developed to explore the influence of genes on intestinal epithelial cell behavior in situ. Complementing the powerful genetics available in the mouse, electroporation provides a rapid method for the analysis of genes that play pivotal roles in intestinal differentiation.
|Pages (from-to)||1779 - 1787|
|Number of pages||9|
|Publication status||Published - 2004|