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Histochemistry and Cell Biology

, Volume 138, Issue 2, pp 201–213 | Cite as

Plasmid DNA is internalized from the apical plasma membrane of the salivary gland epithelium in live animals

  • Monika Sramkova
  • Andrius Masedunskas
  • Roberto WeigertEmail author
Original Paper

Abstract

Non-viral-mediated gene delivery represents an alternative way to express the gene of interest without inducing immune responses or other adverse effects. Understanding the mechanisms by which plasmid DNAs are delivered to the proper target in vivo is a fundamental issue that needs to be addressed in order to design more effective strategies for gene therapy. As a model system, we have used the submandibular salivary glands in live rats and we have recently shown that reporter transgenes can be expressed in different cell populations of the glandular epithelium, depending on the modality of administration of plasmid DNA. Here, by using a combination of immunofluorescence and intravital microscopy, we have explored the relationship between the pattern of transgenes expression and the internalization of plasmid DNA. We found that plasmid DNA is internalized: (1) by all the cells in the salivary gland epithelium, when administered alone, (2) by large ducts, when mixed with empty adenoviral particles, and (3) by acinar cells upon stimulation of compensatory endocytosis. Moreover, we showed that plasmid DNA utilizes different routes of internalization, and evades both the lysosomal degradative pathway and the retrograde pathway towards the Golgi apparatus. This study clearly shows that in vivo approaches have the potential to address fundamental questions on the cellular mechanisms regulating gene delivery.

Keywords

Intravital microscopy Plasmid DNA Endocytosis Salivary glands Gene therapy Non-viral gene delivery 

Notes

Acknowledgments

This research was supported by the Intramural Research Program of the NIH, National Institute of Dental and Craniofacial Research. We would like to thank Drs. Amornphimoltham and Porat-Shliom for critical reading of the manuscript.

Supplementary material

418_2012_959_MOESM1_ESM.pdf (5.8 mb)
Supplementary Fig. 1 Localization of subcellular markers in intact rat submandibular glands. Rat submandibular glands were excised, processed for immunofluorescence, labeled with Phalloidin (green or cyan) and EEA1, Lamp1, TnfR, Vamp4, and GM130, and imaged by confocal microscopy (PDF 5902 kb)
418_2012_959_MOESM2_ESM.mov (9 mb)
The internalization of Rh-pvenus in the presence of rAd5 was imaged by intravital two-photon microscopy (Excitation wavelength 930 nm). Small vesicles (arrow) are internalized from a large duct filled with Rh-pVenus (arrowheads) (MOV 9266 kb)

The trafficking of Rh-pvenus (red) and Alexa 488-Dextran in the presence of rAd5 was imaged by intravital two-photon microscopy after 5 min from the injection of the two probes (Excitation wavelength 930 nm). In the center of the field of view a small vesicle containing Rh-pVenus fuses with a vesicle containing Alexa 488-dextran (arrow) (still images in Fig. 5a). The large duct is highlighted by arrowheads (MOV 1817 kb)

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Copyright information

© Springer-Verlag (outside the USA) 2012

Authors and Affiliations

  • Monika Sramkova
    • 1
  • Andrius Masedunskas
    • 1
  • Roberto Weigert
    • 1
    Email author
  1. 1.Intracellular Membrane Trafficking Unit, Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial ResearchNational Institutes of HealthBethesdaUSA

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