In vivo internalization of plasmid DNA in salivary gland epithelium

Tuesday, October 25, 2011 — Poster Session II
Noon – 2:00 p.m.	Natcher Conference Center	NIDCR	CELLBIO-23

Author

• R Weigert

Abstract

We have previously shown that the various cell populations of the submandibular salivary glands (SMG) can be transfected by the plasmid DNA (pDNA). To determine which endocytic pathway and trafficking steps are involved in the internalization of pDNA we followed the fate of Rhodamine-labeled DNA injected into SMG of live rats. Plasmid DNA injected alone, is internalized by all the epithelial cells in the tissue. However only cells in the intercalated ducts are transfected. Delivery of pDNA with recombinant defective adenoviral particles (rAd5) causes its accumulation in granular convoluted/striated ducts (GC/SD), which are the cell population expressing the gene of interest. When pDNA is delivered during stimulation with the beta-adrenergic agonist isoproterenol, most of the DNA is concentrated in acini, which are transfected. To follow the intracellular route followed by pDNA, we performed immunofluorescent co-staining for different subcellular markers. Interestingly, pDNA was not found associated with the endosomal/lysosomal system at any time, suggesting the early involvement of a mechanism of “endosomal escape”. Ongoing experiments using a pharmacological approach to block the various endocytic pathways will help to determine the machinery utilized by pDNA for the internalization in the SMG epithelium.

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