Efficient gene transfer into the epithelial cell layer of embryonic mouse intestine using low-voltage electroporation.
OBJECTIVE
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.
Abud
Helen E
HE
Ludwig Institute for Cancer Research, PO Box 2008, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia. Helen.abud@ludwig.edu.au
Lock
Peter
P
Heath
Joan K
JK
D000818:Animals; D018274:Electroporation / Q000379:methods*; D004815:Epidermal Growth Factor / Q000502:physiology; D004847:Epithelial Cells; D005260:Female; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D007413:Intestinal Mucosa / Q000166:cytology*; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008813:Mice, Inbred ICR; D011247:Pregnancy; D015398:Signal Transduction
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004815:Epidermal Growth Factor