Background: Transfecting gastrointestinal cells with DNA to express peptide has numerous potential clinical applications in the fields of motility, mucosal ulceration and carcinogenesis. Current methods of transfection are very inefficient. DNA plasmid often fails to traverse the cell cytoplasm to enter the nucleus. The M9 nuclear localization peptide, a fragment of the naturally occurring heterogenous nuclear ribonucleoprotein A1, which serves to shuttle mRNA across the nuclear membrane has been proposed as a tool for enhancing transfection efficiency. (Nature Biotechnology 17,873-877,1999). We tested three different reporter plasmids to assess the ability of M9 to improve transfection efficiency in esophageal mucosal cells.
Methods: The non-malignant esophageal epithelial cell line HET-1a and adenocarcinoma cell line SEG-1 were grown to confluence in DMEM & 10% FCS. Transfection was carried out by incubation for 2 hours at 37oC at a plasmid (±M9) concentration of 1mg/ml and 2mL/ml lipofectamine. Media was removed and cells grown for a further 48hrs in DMEM & 10% FCS, prior to assays. Quantitative b-galactosidase enzyme activity was determined spectrophotometrically KGF-1 was quantitated by ELISA against a KGF-1 standard curve. Green Fluorescent Protein (GFP) expression was assessed by counting the total number of transfected cells per well by fluorescent microscopy.
Results: The M9 nuclear shuttle peptide increased the transfection efficiency (Table 1). b-galactosidase enzyme activity was increased approximately four times using a 50:1 ratio of M9 to plasmid when compared to lipofectamine only transfection (p<0.01 ANOVA). KGF-1 growth factor levels were also increased over 5 fold at a 50:1 ratio (p<0.01 ANOVA). The number of transfected cells expressing GFP was progressively increased with higher M9:plasmid ratios.
Conclusion: The M9 shuttle peptide increases transfection efficiency and therefore may have a useful role in gene therapy applications involving the esophagus.
Table 1: M9-Sct to Plasmid Ratio
no plasmid_ 0:1 10:1 50:1 100:1
b-gal (A570) 0 .268±.06 .338±.007 1.214±.095 -
KgF1 (pg/ml) 0 298±116 284±28 1724±187 -
GFP (cells) 0 196±39 269±36 376±54 690±102