A nuclear targeting peptide, M9, improves transfection efficiency in fibroblasts
| Autor: | Colman K. Byrnes, John W. Harmon, Petra H. Nass, Mark D. Duncan |
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| Rok vydání: | 2002 |
| Předmět: |
animal structures
viruses Heterogeneous Nuclear Ribonucleoprotein A1 Green Fluorescent Proteins Molecular Sequence Data Nuclear Localization Signals Biology Transfection Green fluorescent protein chemistry.chemical_compound Mice Plasmid Heterogeneous-Nuclear Ribonucleoprotein Group A-B Animals Amino Acid Sequence Cation Exchange Resins Fluorescent Dyes Rhodamines fungi Drug Synergism 3T3 Cells Genetic Therapy beta-Galactosidase Molecular biology Lipids Luminescent Proteins chemistry Cytoplasm Cell culture Lipofectamine Surgery Indicators and Reagents Nuclear localization sequence DNA Plasmids |
| Zdroj: | The Journal of surgical research. 108(1) |
| ISSN: | 0022-4804 |
| Popis: | 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 β-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 β-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. |
| Databáze: | OpenAIRE |
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