Evaluation of Viral and Mammalian Promoters for Use in Gene Delivery to Salivary Glands  Changyu Zheng, Bruce J. Baum  Molecular Therapy  Volume 12, Issue 3, Pages 528-536 (September 2005) DOI: 10.1016/j.ymthe.2005.03.008 Copyright © 2005 Terms and Conditions

FIG. 1 Comparison of promoter activities in vitro and in vivo after transfection using plasmid–PEI–Adcontrol complexes. (A) Promoter activities in vitro in A5 cells. Each cell was transfected with 5 × 104 plasmid molecules. See text for details. The data shown are the means ± SD of luciferase activity from three determinations 24 h after transfection and are representative of two separate experiments. For some determinations the error bars are too small to be visualized. (B) Promoter activities in vivo in rat submandibular glands. Each rat submandibular gland was transfected with 4.35 × 1012 plasmid molecules. See text for details. The data shown are the means ± SD of luciferase activity from six submandibular glands of three rats 24 h after transfections. The absence of activity with the K19-0.7 promoter was verified by an additional experiment. Molecular Therapy 2005 12, 528-536DOI: (10.1016/j.ymthe.2005.03.008) Copyright © 2005 Terms and Conditions

FIG. 2 Comparison of promoter activities from eight adenoviral vectors in A5 cells. All adenoviral vectors contained the luciferase cDNA as a reporter gene. Each cell was infected with 100 vector particles. See text for details. The data shown are the means ± SD of luciferase activity from three determinations, 1, 2, or 3 days postinfection, and are representative of two separate experiments. For some determinations the error bars are too small to be visualized. Differences between results (promoter-driven luciferase activity) obtained at individual time points were determined, as appropriate, with a one-way ANOVA or Kruskal–Wallis one-way ANOVA on ranks. Thereafter, determinations of the rank order of promoter activities used the day 3 data and a Kruskal–Wallis one-way ANOVA on ranks, followed by a multiple pair-wise comparison with the Student–Newman–Keuls method, to assess statistical significance (see text for additional details). Molecular Therapy 2005 12, 528-536DOI: (10.1016/j.ymthe.2005.03.008) Copyright © 2005 Terms and Conditions

FIG. 3 Comparison of promoter activities from eight adenoviral vectors in rat submandibular glands. All adenoviral vectors contained the luciferase cDNA as a reporter gene. Each rat submandibular gland was infected with 109 particles. See text for details. The data shown are the means ± SD of luciferase activity from six submandibular glands of three rats at 2, 3, or 7 days postinfection. Differences between results (promoter-driven luciferase activity) obtained at individual time points were determined, as appropriate, with a one-way ANOVA or Kruskal–Wallis one-way ANOVA on ranks. Thereafter, determinations of the rank order of promoter activities used the day 3 data and a Kruskal–Wallis one-way ANOVA on ranks, followed by a multiple pair-wise comparison with the Student–Newman–Keuls method, to assess statistical significance (see text for additional details). Molecular Therapy 2005 12, 528-536DOI: (10.1016/j.ymthe.2005.03.008) Copyright © 2005 Terms and Conditions

FIG. 4 Comparison of promoter activities in A5 cells and rat submandibular glands using adenoviral vectors encoding EGFP as a reporter gene. (A) Results with A5 cells. Fluorescence microscopy was performed 48 h after infection. Each cell was infected with 100 vector particles. This experiment was repeated three separate times with similar results. Pictures in the top row were taken at a low sensitivity setting (Max 200). Pictures in the middle row were taken at an intermediate sensitivity setting (Max 12). Pictures in the bottom row were taken at a higher sensitivity setting (Max 2). (B, C) Results with rat submandibular glands. Rat submandibular glands were infected with 109 vector particles/gland using one of the following promoters: CMV, RSV, EF1α, K18-2.5, or K19-3.0. For experiments evaluating the rAQP5-0.4, Kall, and AMY promoters, 1010 particles/gland were delivered. (B) Hematoxylin and eosin staining of tissue samples from a control gland (no viral infection) and a gland following infection with AdAMY-EGFP. (C) Immunohistochemical staining using an antibody directed at EGFP. Results are representative of four submandibular glands from two rats, 3 days after vector administration, with three sections from each gland examined. In individual images, “a” indicates acinar cells and “d” indicates ductal cells. Molecular Therapy 2005 12, 528-536DOI: (10.1016/j.ymthe.2005.03.008) Copyright © 2005 Terms and Conditions

Supplementary Figure 1. The plasmid, pAC-luc Supplementary Figure 1. The plasmid, pAC-luc. This plasmid was constructed using pACCMV-pLpA that contains part of the adenovirus serotype 5 genome (0–1.2 and 9.2–17 mu). An 1851 bp luciferase cDNA was used as a reporter gene, along with a 307 bp multiple cloning site and SV40 poly A sequence, all placed between map units 1.3 and 9.1. pAC-luc also serves as a shuttle vector for the generation of recombinant serotype 5, E1− adenoviral vectors. See Materials and Methods for additional details. Molecular Therapy 2005 12, 528-536DOI: (10.1016/j.ymthe.2005.03.008) Copyright © 2005 Terms and Conditions