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

Abstract

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.

Role for the actomyosin complex in regulated exocytosis revealed by intravital microscopy

“…Next, we analyzed the dynamics of F-actin recruitment onto the SCGs by transfecting the acini of live rats with GFP-lifeact, a probe that dynamically labels F-actin without compromising its function (37). We have previously shown that the acini in the SGs can be selectively transfected with fluorescently tagged proteins (27). As expected, in acinar cells, GFP-lifeact showed a distribution comparable to that of F-actin.…”

“…Although we have successfully analyzed the dynamics of large SCGs (this study) and endosomal structure (27)(28)(29), it will probably be more difficult to study smaller structures such as 50-nm synaptic vesicles that exocytose in a few milliseconds. However, microscopes with faster scanning speed and genetically encoded probes with higher quantum efficiency may soon overcome this current difficulty.…”

“…First, we slowly infused a fluorescently labeled dex- tran into the ductal system of a live rat (Fig. 2D) (27). This approach mimics the bathing of the explanted glands in small fluorescent probes that has been used to investigate exocytosis in ex vivo models (11,13,14,16,17,35).…”

“…Our approach used confocal intravital microscopy (IVM) as the imaging technique and the submandibular SGs of live rodents, a model organ that we have previously used to study membrane trafficking under physiological conditions (25)(26)(27).…”

Human Parathyroid Hormone Is Secreted Primarily into the Bloodstream After Rat Parotid Gland Gene Transfer

“…The present study has demonstrated that there is a difference in how transgenic hPTH is routed intracellularly between rat submandibular and parotid glands, and ultimately secreted. Using hPTH as a model protein to study the sorting pathways operative in these two exocrine glands should lead to a better understanding of how transgenic proteins are handled in salivary epithelial cells and, presumably, result in the identification of the key intracellular molecules involved (Baum, 2009;Sramkova et al, 2009). The latter, along with large animal studies, likely would permit a reasonable assessment of the potential to find similar endocrine sorting of transgenic hPTH in human parotid glands as seen herein with the rat parotid gland.…”