Gastroenterology

Gastroenterology

Volume 126, Issue 7, June 2004, Pages 1779-1787
Gastroenterology

Basic-alimentary tract
Efficient gene transfer into the epithelial cell layer of embryonic mouse intestine using low-voltage electroporation

https://doi.org/10.1053/j.gastro.2004.03.006Get rights and content

Abstract

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 β-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 β-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.

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.

References (42)

  • A. Choidas et al.

    The suitability and application of a GFP-actin fusion protein for long-term imaging of the organization and dynamics of the cytoskeleton in mammalian cells

    Eur J Cell Biol

    (1998)
  • P. Lock et al.

    Independent SH2-binding sites mediate interaction of Dok-related protein with RasGTPaseactivating protein and Nck

    J Biol Chem

    (1999)
  • H. Lickert et al.

    Formation of multiple hearts in mice following deletion of beta-catenin in the embryonic endoderm

    Dev Cell

    (2002)
  • J. Huelsken et al.

    β-Catenin controls hair follicle morphogenesis and stem cell differentiation in the skin

    Cell

    (2001)
  • E. Sancho et al.

    Live and let die in the intestinal epithelium

    Curr Opin Cell Biol

    (2003)
  • D.Y. Stainier

    A glimpse into the molecular entrails of endoderm formation

    Genes Dev

    (2002)
  • Q. Yang et al.

    Requirement of Math1 for secretory cell lineage commitment in the mouse intestine

    Science

    (2001)
  • L. van der Weyden et al.

    Tools for targeted manipulation of the mouse genome

    Physiol Genomics

    (2002)
  • R.P. Misra et al.

    Gene targeting in the mouseadvances in introduction of transgenes into the genome by homologous recombination

    Endocrine

    (2002)
  • C.N. Johnstone et al.

    Characterization of mouse A33 antigen, a definitive marker for basolateral surfaces of intestinal epithelial cells

    Am J Physiol

    (2000)
  • N. Itasaki et al.

    “Shocking” developments in chick embryologyelectroporation and in ovo gene expression

    Nat Cell Biol

    (1999)
  • Cited by (15)

    • Stem cell niche: Microenvironment and beyond

      2008, Journal of Biological Chemistry
    • Proteasome modulating agents induce rAAV2-mediated transgene expression in human intestinal epithelial cells

      2005, Biochemical and Biophysical Research Communications
      Citation 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].

    • Regulation of intestinal stem cells by Wnt and Notch signalling

      2013, Advances in Experimental Medicine and Biology
    View all citing articles on Scopus

    Supported in part by National Heath and Medical Research, Australia (grant 191502), awarded to H.E.A. and J.K.H.

    View full text