Basic-alimentary tractEfficient gene transfer into the epithelial cell layer of embryonic mouse intestine using low-voltage electroporation☆
Section snippets
Plasmids
Reporter genes were cloned downstream of the elongation factor 1 α (EF1α) promoter in the pEF/myc/cyto vector (Invitrogen, Carlsbad, CA). A 757-bp SalI-Not1 fragment encoding the EGFP protein and a 1.8-kb fragment encoding the Cre-EGFP fusion protein was inserted into the SalI-NotI site of pEF/myc/cyto to form EF1α-EGFP and EF1α-Cre-EGFP, respectively. These constructs were gifts from M. Faux, M. Condron, and J. Ross. EGFP Cdx2 was constructed by using pEGFP-N1 (Clontech) and was a gift from C.
Defining electroporation conditions
Embryonic gut was harvested from E13.5 and E14.5 embryos to determine the optimal conditions of low-voltage electroporation to produce expression of introduced constructs. At this stage of development the endoderm cell layer consists of a pseudostratified epithelium and the gut lumen is of a sufficient diameter to be perfused with a solution containing plasmid DNA. To analyze the efficiency of electroporation, a plasmid encoding EGFP under the control of the EF1α promoter was injected into the
Discussion
Our results indicate that low-voltage electroporation is an extremely effective technique for manipulating gene expression in embryonic gut explants. This method is rapid and efficient and the explanted material is accessible for observation and experimental manipulation. The epithelial cell layer was easy to target because the plasmid DNA could be placed in the lumen and the negatively charged DNA could be directed to one side of the explant toward the positive electrode. After determination
Acknowledgements
The authors would like to thank Maree Faux, Melanie Condron, Janine Ross (Ludwig Institute, Melbourne), Claire Domon-Dell, and Jean-Paul Freud (INSERM, Strasbourg) for the gifts of constructs; Stacey I for assistance in construct preparation; Natasha Smith for genotyping of the R26R animals; and Gary Hime for assistance with microscopy, imaging, and critical reading of the manuscript.
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2005, Biochemical and Biophysical Research CommunicationsCitation Excerpt :The promise of intestinal gene therapy was demonstrated in vivo by the pioneering work of Henning and co-workers [32] describing retrovirus-mediated transgene delivery to rat intestine. Thus far, many approaches for intestinal gene transfer, including viral vector systems (retrovirus, adenovirus, adeno-associated virus, and lentivirus) [3,8,33–37] and non-viral systems (electroporation and liposome) [38,39], have been tested in animals and in human cellular models. While developing these intestinal gene delivery methods, it has been recognized that mucolytic agents such as dithiothreitol and N-acetyl-cysteine are capable of improving the access of intestinal epithelial cells to vectors [33,40].
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Supported in part by National Heath and Medical Research, Australia (grant 191502), awarded to H.E.A. and J.K.H.