Electroporation enhances transfection efficiency in murine cutaneous wounds.

Transfection of wounds with DNA-encoding growth factors has the potential to improve healing, but current means of nonviral gene delivery are inefficient. Repeated high doses of DNA, necessary to achieve reliable gene expression, are detrimental to healing. We assessed the ability of in vivo electroporation to enhance gene expression. Full-thickness cutaneous excisional wounds were created on the dorsum of female mice. A luciferase- encoding plasmid driven by a CMV promoter was injected at the wound border. Following plasmid administration, electroporative pulses were applied to injection sites. Pulse parameters were varied over a range of voltage, duration, and number. Animals were euthanized at intervals after transfection and the luciferase activity measured. Application of electric pulses consistently increased luciferase expression. The electroporative effect was most marked at a plasmid dose of 50 micro g, where an approximate tenfold increase was seen. Six 100- micro s-duration pulses of 1750 V/cm were found to be the most effective in increasing luciferase activity. High numbers of pulses tended to be less effective than smaller numbers. This optimal electroporation regimen had no detrimental effect on wound healing. We conclude that electroporation increases the efficiency of transgene expression and may have a role in gene therapy to enhance wound healing.

Duodenal reflux produces hyperproliferative epithelial esophagitis--a possible precursor to esophageal adenocarcinoma in the rat.

Esophageal reflux of duodenal contents converts a rat nitrosamine esophageal cancer model from squamous cell carcinoma to adenocarcinoma. Further, there was a tendency for male rats to have a higher incidence of cancer than female rats. However, chemical castration with the gonadotropin-releasing hormone analog leuprolide did not protect male or female animals from developing cancer. We have identified an early (6-week) hyperproliferative epithelial cell reaction to duodenal reflux. We carried out experiments to assess the specificity of duodenal reflux in producing the hyperproliferative epithelial precursor lesion. Animals underwent specific surgical procedures to produce esophageal reflux of pure duodenal contents, mixed gastroduodenal, or bland intestinal contents. A hyperproliferative mucosal esophagitis developed in the group with duodenal reflux but not in the other groups. Mucosal thickness in the duodenal reflux group reached seven times that of normal mucosa at 6 weeks. These results suggest that esophageal reflux of duodenal contents plays an important role in the pathogenicity of proliferative esophagitis and the potential development of esophageal adenocarcinoma.

A nuclear targeting peptide, M9, improves transfection efficiency in fibroblasts.

BACKGROUND: Nonviral transfection of eukaryotic cells remains inefficient. Liposomes can transport DNA plasmid into the cytoplasm, but the nuclear membrane remains a barrier to efficient plasmid DNA transfection. But normal cells have mechanisms to transport nucleic acids across the nuclear membrane. Cells routinely utilize a transporter to carry mRNA from nucleus to cytoplasm. MATERIALS AND METHODS: We used a modified mRNA transporter, the M9 component of heterogeneous nuclear ribonucleoprotein-A1, complexed to a DNA carrier to facilitate DNA transfer into the nucleus. We examined the effect of M9 on transfection in 3T3 fibroblasts. Our hypothesis was that the M9 shuttle would increase transfection efficiency by delivering plasmid to the nucleus, after cytoplasmic entry was facilitated by Lipofectamine. Transfection was assessed using plasmids expressing beta-galactosidase and green fluorescent protein (GFP). Intracellular location of rhodamine-labeled plasmid was determined by fluoroscopic microscopy. RESULTS: In the fluorescent microscopy experiments, we found that rhodamine-labeled DNA plasmid was sequestered in the cytoplasm in the Lipofectamine-treated cells, but gained access to the nucleus with the addition of M9. At concentrations where neither M9 nor Lipofectamine individually increased plasmid mediated transfection, as evidenced by beta-galactosidase activity; their combination increased transfection dramatically by approximately 20-fold, from 2 +/- 1 to 32 +/- 5. CONCLUSIONS: As expected, based on their presumed actions, Lipofectamine and the M9 shuttle synergistically promote efficient cellular transfection. Efficient cellular transfection will be required in clinical applications of gene therapy.

Novel nuclear shuttle peptide to increase transfection efficiency in esophageal mucosal cells.

The major barrier to successful transfection appears to be passage of the DNA plasmid from the cytoplasm into the cell nucleus. The M9 nuclear localization peptide, a fragment of the naturally occurring heterogeneous nuclear ribonucleoprotein A1, which serves to shuttle messenger RNA across the nuclear membrane, has been proposed as a tool for enhancing transfection efficiency. We tested three different reporter plasmids to assess the ability of M9 to improve transfection efficiency in esophageal mucosal cells. The effect of M9 on the intracellular movement of plasmid was also assessed using fluorescent microscopy to trace rhodamine-labeled plasmid. The M9 nuclear shuttle peptide consistently increased the transfection efficiency. When transfection was carried out with specific plasmids, beta-galactosidase enzyme activity, keratinocyte growth factor-1 growth factor levels, and the number of transfected cells expressing growth factor peptides were progressively increased with increasing M9 to plasmid ratios. Fluorescent microscopy demonstrated that the M9 shuttle allowed rhodamine-tagged plasmid to gain access to the nucleus, while it was located exclusively in the cytoplasm without the peptide. The M9 shuttle peptide increases transfection efficiency in esophageal mucosal cells, and therefore may have a useful role in gene therapy applications involving the esophagus.