Efficient gene transfer into the epithelial cell layer of embryonic mouse intestine using low-voltage electroporation  Helen E. Abud, Peter Lock, Joan K. Heath  Gastroenterology  Volume 126, Issue 7, Pages 1779-1787 (June 2004) DOI: 10.1053/j.gastro.2004.03.006

Figure 1 Electroporation of embryonic gut explants. (A) Schematic representation of the electroporation procedure. DNA is injected into the lumen of E13.5–E14.5 of embryonic gut and electric pulses are applied across the gut segment by using wire electrodes. The DNA is specifically electroporated into one side of the endoderm/epithelial cell layer. (B) Effect of voltage on EGFP expression rates in embryonic gut explants. The percentage of explants containing EGFP-positive cells was assessed. Explants were scored positive if they contained >1% positive epithelial cells when viewed under the dissecting microscope. Data were collected from at least 5 separate experiments for each voltage level (3 pulses were applied at each voltage level). Error bars indicate SD. Gastroenterology 2004 126, 1779-1787DOI: (10.1053/j.gastro.2004.03.006)

Figure 2 EGFP expression in the epithelial cell layer of electroporated gut explants. All panels depict E13.5 embryonic gut explants 48 hours after electroporation with α-EGFP plasmid DNA. (A—E ) Images of live gut, (F—H ) fixed tissue. (A, C) Bright-field image of E13.5 explant attached to filter paper support, (B, D) fluorescent images of A and C, respectively, showing many cells expressing EGFP on one side of the epithelium. (E ) Confocal optical section of endoderm cell layer (longitudinal). (F ) Confocal optical section of the epithelial cell layer of explant tissue imaged from the luminal surface and costained with Sytox Orange to highlight nuclei. (G) Transverse section of gut explant processed for immunohistochemistry with anti-GFP Alexa 488. (H ) Higher magnification of G. (G, H) Brackets indicate the width of the electric field. Gastroenterology 2004 126, 1779-1787DOI: (10.1053/j.gastro.2004.03.006)

Figure 3 Cellular localization and stability of expression of EGFP fusion proteins. (A, B) E13.5 embryonic gut explants 48 hours after electroporation. (C—E ) Tissue that was electroporated with EF1α-EGFP at E13.5, placed into catenary culture for 2 days, and transplanted under the kidney capsule of host mice for 2 weeks. (A ) Confocal maximum intensity projection (extended focus image) of a live gut explant electroporated with EGFP actin. Four electroporated cells show the formation of an actin ring at their apical surface. (B) Confocal optical section of a fixed gut explant electroporated with EGFP-Cdx2 (green nucleus) and processed for immunohistochemistry for A33 antigen to outline the cells.10 (C ) Whole-mount image of kidney capsule graft showing the presence of EGFP-expressing cells (indicated by arrow). (D) Section of kidney capsule graft depicted in C, stained with H&E. (E ) Section of kidney capsule graft stained with alcian blue/periodic acid-Schiff to highlight mucin-secreting goblet cells (stained purple and indicated by arrows). H, host kidney tissue. Gastroenterology 2004 126, 1779-1787DOI: (10.1053/j.gastro.2004.03.006)

Figure 4 Monitoring changes in the localization of EGFP fusion proteins in live cells. E14.5 embryonic gut was electroporated with plasmids encoding (A, B) DokR-EGFP or (C, D) DokR-EGFP48. After 24 hours in culture, individual cells within the live explants were imaged from the luminal surface at high magnification, (A, C) in the absence of EGF stimulation or (B, D) after 5 minutes of stimulation with EGF, 10 ng/mL. Gastroenterology 2004 126, 1779-1787DOI: (10.1053/j.gastro.2004.03.006)

Figure 5 Functional activity of Cre recombinase gene electroporated into gut explants. Embryonic gut was collected from (A—E ) R26R and (F ) wild-type embryos at E13.5, electroporated, and placed in catenary culture for 24 hours before fixation and staining for β-galactosidase activity. All panels were photographed using bright-field microscopy except for C, which was visualized with phase-contrast microscopy. (A—C, F) Explants were electroporated with Cre-EGFP plasmid DNA. (A ) Midgut, (B) hindgut, (C ) hindgut, (D) nonelectroporated control, (E ) EGFP plasmid DNA electroporation, (F ) Cre-EGFP electroporation into wild-type tissue. Gastroenterology 2004 126, 1779-1787DOI: (10.1053/j.gastro.2004.03.006)