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.
Byrnes
Colman K
CK
Department of Surgery, Queens University, Belfast, UK.
Nass
Petra H
PH
Shim
Joon
J
Duncan
Mark D
MD
Lacy
Brian
B
Harmon
John W
JW
D000704:Analysis of Variance; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000378:metabolism*; D004247:DNA / Q000235:genetics*; D004947:Esophagus / Q000166:cytology; D051523:Fibroblast Growth Factor 7; D005346:Fibroblast Growth Factors / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence; D009092:Mucous Membrane / Q000166:cytology; D010957:Plasmids; D011336:Probability; D021382:Protein Sorting Signals; D012680:Sensitivity and Specificity; D014162:Transfection
C496453:FGF7 protein, human; D021382:Protein Sorting Signals; D051523:Fibroblast Growth Factor 7; D005346:Fibroblast Growth Factors; D004247:DNA