Figure 5: PCR analysis for detection and localization of lentiviral vector within canine genome.

(a) Long-term detection of BDDFVIII-lentiviral vector within leukocyte genomic DNA. Shown is an ethidium bromide-stained agarose gel of PCR product-derived genomic DNA isolated from canine peripheral blood leukocytes. Molecular weight markers (Lane 1) are labelled on the left in base pairs (bp). In Lane 2, p−889ITGA2B-BDDFVIII-WPTS served as a positive control for PCR of a 302 bp region of the lentiviral WPRE. PCR failed to detect WPRE within leukocyte genomic DNA from a FVIII-deficient dog serving as a negative control (Lane 3). In contrast, WPRE was detected in the PCR of all three experimental dogs (F20, I42, M64; Lanes 4–6) for at least 2.5 years after G-PBC transplant demonstrating long-term insertion of the lentiviral vector into the canine genome. All reactions were run in triplicate from at least three separate samples collected periodically for at least 2.5 years after G-PBC transplant. (b) Linear amplification-mediated (LAM)-PCR to localize lentiviral vector within canine genome. Polyacrylamide gel of LAM-PCR products from a FVIII-deficient negative control and experimental dogs at 2.5 (F20), 1.4 (I42) and 0.5 (M64) years after infusion of FVIII-transduced G-PBC. PCR was performed on genomic DNA isolated from leukocytes purified with Ficoll from circulating peripheral blood. Each amplified band represents a distinct proviral integration. All lanes appear to contain multiple insertions, indicative of polyclonality. The panel underwent black–white inversion from the original digital image, and levels were globally adjusted to improve visibility of all bands. DNA standard markers (Lane 1 and 6) labelled on left in base pairs.