G box hr

The AAV8 vector expressing human secreted embryonic alkaline phosphatase (AAV8-huSEAP) and the AAV8-U7 vector^{49 (link)} were produced in a baculovirus system^{50 (link)}. The AAV8 vector encoding for human factor IX (AAV8-hF.IX) was previously described^{37 (link)}, this vector and the preparations of full and empty vector particles were made as previously described⁵¹ . All vectors were titrated using a quantitative real-time PCR assay. Empty AAV capsids were quantified using SDS-PAGE after Sypro Ruby staining (Molecular Probes, Thermo Fisher Scientific, Waltham, MA). Gel images were acquired on a gel reader G:box HR (Syngene, Frederick, MD) and the bands corresponding to the VP3 capsid protein quantified against a standard of known concentration using the software Image studio light (LI-COR Biosciences, Lincoln, NE).

PCR products (0.2‒0.5 μg) were cut in a final volume of 30 μL by the restriction endonuclease enzymes FokI, ApaI, TaqI, and BsmI (Thermo Fisher Scientific, USA) for 16 hours. The digestion products were run on 2% agarose gel to determine the number and length of bands. The gels were stained with GelRed (Biotium, Belgium) and photographed with G:BOX HR (Syngene, Cambridge, UK).