Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Suppression of leaky expression of adenovirus genes by insertion of microRNA-targeted sequences in the replication-incompetent adenovirus vector genome

doi: 10.1038/mtm.2014.35

Figure Lengend Snippet: Ad vector-induced immune responses following intravenous administration of Ad vectors into mice. ( a ) Hexon-specific IFN-γ + CD8 + T cells in the splenocytes. C57BL/6 mice were intravenously administered Ad vectors at 1 × 10 10 IFU/mouse. Fifteen days after the injection, the splenocytes were harvested. The splenocytes were incubated with hexon peptide for 6 hours. ( b ) Infiltration of lymphocytes into the liver following Ad vector administration. C57BL/6 mice were intravenously administered Ad vectors at 1 × 10 10 IFU/mouse. Ten days after administration, liver mononuclear cells were isolated and analyzed for cells expressing CD3, CD4, and CD8 by fluorocytometry. The data are expressed as the mean values ± SD ( n = 6). N. S., not significant. ( c ) IFN-γ and chemokine mRNA levels in the liver after administration of Ad-L2 and Ad-E4-122aT-L2. C57BL/6 mice were treated with Ad vectors at 1 × 10 10 IFU/mouse. Ten days after administration, IFN-γ and chemokine mRNA levels in the liver were determined by real-time RT-PCR. The data are expressed as the mean values ± SD ( n = 3–6). ( d ) Anti-Ad antibody levels in the serum following intravenous administration of Ad vectors. C57BL/6 mice were intravenously administered as described above. Anti-Ad antibody levels in the serum were determined by ELISA 14 days after administration. The data are expressed as the mean values ± SD ( n = 6). ( e ) Ad-specific CTL-mediated lysis of the hepatocytes transduced with Ad vectors. C57BL/6 mice were intravenously administered Ad-null at a dose of 1 × 10 10 IFU/mouse. Ten days after the injection, the splenocytes were harvested and incubated with Ad-null at an MOI of 10 for 4 days. Primary mouse hepatocytes were transduced with Ad-L2 or Ad-E4-122aT-L2 at an MOI of 10 for 24 hours and were incubated with the splenocytes at 37 °C. LDH levels in the medium were measured 4 hours after incubation. The data are expressed as the mean values ± SD ( n = 4). * P < 0.05 in comparison with Ad-L2.

Article Snippet: Finally, the plate was washed with PBST and TMB ELISA Peroxidase Substrate (Rockland Immunochemicals, Gilbertsville, PA) was added.

Techniques: Plasmid Preparation, Injection, Incubation, Isolation, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Lysis, Transduction, Comparison