Expression of plasmid DNA in the salivary gland epithelium: novel approaches to study dynamic cellular processes in live animals.
The ability to dynamically image cellular and subcellular structures in a live animal and to target genes to a specific cell population in a living tissue provides a unique tool to address many biological questions in the proper physiological context. Here, we describe a powerful approach that is based on the use of rat submandibular salivary glands, which offer the possibility to easily perform intravital imaging and deliver molecules from the oral cavity, and plasmid DNA, which offers the advantage of rapid manipulations. We show that, under different experimental conditions, a reporter molecule can be rapidly expressed in specific compartments of the glands: 1) in the intercalated ducts, when plasmid DNA is administered alone, and 2) in granular ducts, striated ducts, and, to a lesser extent, acini, when plasmid DNA is mixed with replication-deficient adenovirus subtype 5 particles. Remarkably, we also found that gene expression can be directed to acinar cells when plasmid DNA is administered during isoproterenol-stimulated exocytosis, suggesting a novel mechanism of plasmid internalization regulated by compensatory endocytosis. Finally, as a practical application of these strategies, we show how the expression of fluorescently tagged molecules enables the study of the dynamics of various organelles in live animals at a resolution comparable to that achieved in cell cultures.
Intracellular Membrane Trafficking Unit, National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD 20892-4340, USA.
Monika
M
Sramkova
Andrius
A
Masedunskas
Laura
L
Parente
Alfredo
A
Molinolo
Roberto
R
Weigert
D000222:Adaptation, Physiological; D000256:Adenoviridae / Q000145:classification; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002468:Cell Physiological Phenomena; D004247:DNA / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology; D004848:Epithelium / Q000378:metabolism / Q000821:virology; D005089:Exocytosis / Q000187:drug effects; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D042541:Intracellular Space / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D010827:Physiology / Q000379:methods* / Q000639:trends*; D010957:Plasmids / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013363:Submandibular Gland / Q000166:cytology / Q000378:metabolism* / Q000502:physiology / Q000821:virology; D014018:Tissue Distribution; D014771:Virion; D014779:Virus Replication
D015415:Biomarkers; D005456:Fluorescent Dyes; D004247:DNA; D007545:Isoproterenol