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.


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.
Byrnes
Colman K
CK
Johns Hopkins Medical Institutions, Baltimore, Maryland 21224, USA.
Nass
Petra H
PH
Duncan
Mark D
MD
Harmon
John W
JW
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002411:Cation Exchange Resins / Q000494:pharmacology; D004357:Drug Synergism; D005456:Fluorescent Dyes; D015316:Genetic Therapy / Q000379:methods; D049452:Green Fluorescent Proteins; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000235:genetics / Q000493:pharmacokinetics*; D007202:Indicators and Reagents / Q000378:metabolism; D008055:Lipids / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D010957:Plasmids / Q000493:pharmacokinetics; D012235:Rhodamines; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics
D002411:Cation Exchange Resins; D005456:Fluorescent Dyes; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; C000619657:Hnrnpa1 protein, mouse; D007202:Indicators and Reagents; D008055:Lipids; C086724:Lipofectamine; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D012235:Rhodamines; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase