Proparacaine hydrochloride ophthalmic solution

AAV was diluted into 100 µl of sterile 0.9% NaCl USP (Fresenius Kabi, 918610) before injection. Anaesthesia was induced with 2–4% isoflurane. Following induction, as measured by unresponsiveness to bilateral toe pinch, the right eye was protruded by gentle pressure on the skin, and an insulin syringe was advanced, with the bevel facing away from the eye, into the retrobulbar sinus where AAV solution was slowly injected. One drop of proparacaine hydrochloride ophthalmic solution (Patterson Veterinary, 07–885–9765) was then applied to the eye as an analgesic. At collection, mice were euthanized by carbon dioxide asphyxiation. Genomic DNA was purified from minced tissue using gDNAdvance kit (Beckman Coulter, A48705) according to the manufacturer’s instructions and used as template for high-throughput sequencing. RNA was purified from 30 mg of snap-frozen liver tissue with RNeasy Plus mini kit (Qiagen 74134) according to the manufacturer’s instructions, then reverse transcribed to cDNA using SuperScript III first-strand synthesis supermix (Invitrogen, 18080–450) with an oligo dT primer, which was used as template for high-throughput sequencing.

50 μL of VLPs (containing 4x10¹¹ or 7x10¹¹ VLPs) were centrifuged for 10 min at 15,000 g to remove debris. The clarified supernatant was diluted to 120 μL in 0.9% NaCl (Fresenius Kabi; 918610) right before injection. 1x10¹¹ viral genomes (vg) of total AAV was diluted to 120 μL in 0.9% NaCl (Fresenius Kabi; 918610) right before injection. Anesthesia was induced with 4% isoflurane. Following induction, as measured by unresponsiveness to bilateral toe pinch, the right eye was protruded by gentle pressure on the skin, and an insulin syringe was advanced, with the bevel facing away from the eye, into the retrobulbar sinus where VLP or AAV mix was slowly injected. One drop of Proparacaine Hydrochloride Ophthalmic Solution (Patterson Veterinary; 07-885-9765) was then applied to the eye as an analgesic. Genomic DNA was purified from various tissue using Agencourt DNAdvance kits (Beckman Coulter; A48705) following the manufacturer’s instructions.