Rheb(S16H) AAV1 Gene Delivery Protocol

All vectors used in these studies were the AAV1 serotype as previously described [10 (link), 14 (link), 15 (link)]. A plasmid carrying Rheb was purchased from OriGene Technologies (Rockville, MD, USA). Rheb DNA was amplified and modified to incorporate a FLAG-encoding sequence at the 3'-end by expanded long-template PCR (Roche, Basel, Switzerland). Rheb(S16H) was generated with the Phusion Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA) in the pGEM-T vector (Promega, Madison, WI, USA), and then cloned into an AAV packaging construct that utilizes the chicken β-actin promoter and contains a 3' WPRE (pBL). AAVs were produced by the University of North Carolina Vector Core, and the genomic titer of Rheb(S16H) was 2×10¹² viral genomes/ml. Enhanced green fluorescent protein (GFP), used as a control, was subcloned into the same viral backbone, and viral stocks were produced at titers of 1×10¹² viral genomes/ml.