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Research Article

Expression of plasmid DNA in the salivary gland epithelium: novel approaches to study dynamic cellular processes in live animals

¹NIDCR/NIH

Submitted 18 June 2009 ; revised 24 September 2009 ; accepted in final form 24 September 2009

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 based on the use of both rat submandibular salivary glands, which offers the possibility to easily perform intravital imaging and deliver molecules from the oral cavity, and plasmid DNA, offering 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: i) in the intercalated ducts, when plasmid DNA is administered alone, and ii) in granular ducts, striated ducts and to a lesser extent in acini, when plasmid DNA is mixed with replication deficient adenovirus subtype 5 (rAd5) particles. Remarkably, we also found that gene expression can be directed to acinar cells, when plasmid DNA is administered while exocytosis is being stimulated with isoproterenol, 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

Salivary Glands; Gene tranduction; Plasmid DNA; Intravital two-photon microscopy; Compensatory Endocytosis