Viral vectors were generated by triple transfection of HEK293 cells and purified as previously described (Matsushita et al., 1998 (link); Ayuso et al., 2010 (link)). Cells were cultured in roller bottles (Corning) in DMEM supplemented with 10% FBS to 80% confluence and cotransfected with a plasmid carrying the expression cassette flanked by the AAV2 viral inverted terminal repeats, a helper plasmid carrying the AAV rep2 and cap9 genes, and a plasmid carrying the adenovirus helper functions (plasmids kindly provided by K.A. High, Children’s Hospital of Philadelphia, Philadelphia, PA). The expression cassettes were under the control of the cytomegalovirus promoter and contained a SV40 polyA signal for eGFP, and the cytomegalovirus promoter and the 3′-untranslated region of the Tert gene as polyA signal for Tert. AAV9 particles were purified using two cesium chloride gradients, dialyzed against PBS, filtered, and stored at −80°C until use. Then, 27–30-wk old Tert+/+ and G3 Tert−/− mice were administered with 2E12vg of AAV9-Tert and AAV9-empty virus particles in a volume of 100 µl 0.001% Pluronic F-68 in PBS 1X by a single intravenous tail injection (Fig. 6 A). Pluronic F-68 was used to prevent aggregations of the virus particles (Foust et al., 2009 (link)). Animals were then sacrificed 21 wk after the administration of the AAV9-Tert and AAV9-empty virus particles.