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. Author manuscript; available in PMC: 2017 Jan 25.
Published in final edited form as: Mol Ther. 2008 Mar 25;16(6):1089–1097. doi: 10.1038/mt.2008.56

Figure 5. Assessment of vector DNA in situ.

Figure 5

(a) Diagram of PCR products amplified with AdLTR2EF1α-hEPO. The PCR1 amplicon goes from the beginning of the upstream Moloney murine leukemia virus element to the middle of the EF1α promoter. The PCR2 amplicon goes from the EF1α promoter to the middle of the hEPO cDNA. The EPO amplicon is from the middle of the hEPO cDNA. The PCR3 amplicon goes from the hEPO cDNA into the Ad5 E2 region. The PCR4 amplicon is at the 3′-end of the vector in the Ad5 E4 region. (b) Detection of the EPO amplicon in targeted glands 6 months after vector delivery. Note that vectors were delivered to the right (R) submandibular glands of all animals except rat #5 in the AdLTR2EF1α-hEPO group, which received the vector in the left (L) gland. (c) Detection of the amplicons PCR 1, 2, and 3 in targeted glands 1 year after AdLTR2EF1α-hEPO delivery to female and male rats. (d) Detection of the amplicons PCR 1, 2, and 3 in targeted glands 6 months after AdLTR2EF1α-hEPO delivery, and PCR 4 after delivery of AdLTR2EF1α-hEPO (LTREPO), AdEF1α-hEPO (EF1αEPO), and AdCMV-hEPO (CMVEPO). (e) G418-resistant colonies obtained from A5 cells transduced with either AdEF1α-neo or AdLTR2EF1α-neo (neo, neomycin resistance gene). (f) PCR assays (using EF1αF4 and neoB3 primers) with high-molecular-weight (HMW) and low-molecular-weight (LMW) DNA extracts from G418-resistant colonies. (g) Assay for the PCR2 amplicon using HMW and LMW DNA extracts from female rats 1 year after administration of either AdEF1α-hEPO or AdLTR2EF1α-hEPO. (h) Plasmid-safe DNase assays to determine the general vector DNA structure (linear double strand or circular) in rats whose submandibular glands had been transduced with either AdLTR2EF1α-hEPO or AdEF1α-hEPO 1year earlier. Plus means presence of plasmid-safe DNase in the incubation mixture, Minus means without plasmid-safe DNase present. (i) Southern hybridization of undigested DNA samples from targeted glands 6 months after delivery of AdLTR2EF1α-hEPO, AdEF1α-hEPO, or AdCMV-hEPO. P refers to positive control samples from intact vectors spiked into glands from naive rats during genomic DNA extraction. Ad, adenovirus; CMV, cytomegalovirus; hEF1α, human elongation factor-1α; EtBr, ethidium bromide; hEPO, human erythropoietin; LTR, long-terminal repeat.