2v6.11 cells

HEK293 cells purchased from Agilent Technologies (Santa Clara, CA, USA) and 2V6.11 cells obtained from ATCC (Manassas, VA, USA) were maintained as described previously [24 (link)].
The AAV8 vector encodes the luciferase gene located downstream of the liver-specific chimeric promoter. It also contains an enhancer element from the hepatic control region (HCR) of the ApoE/C-I gene and the 5′ flanking region of the human α 1-antitrypsin (HAAT) gene (AAV8-HCRHAAT-luciferase), as described previously [32 (link)]. The AAV vector was prepared with the chimeric packaging plasmid (AAV2 rep/AAV8 cap) and the adenovirus helper plasmid, pHelper (Agilent Technologies), as described previously [33 (link)]. Titration of the recombinant AAV vectors was performed by quantitative PCR using the real-time PCR system StepOnePlus™ (Applied Biosystems, Tokyo, Japan), as described previously [34 (link)].

The highly AAV-permissive 2V6.11 cells (CRL-2784, ATCC, Manassas, VA, USA) [19 (link)] were plated at a density of 2 × 10⁵ cells/well in the presence of ponesterone at 1 µg/mL in a 24 well plate. The next day, cells were counted and based on the cell density; rAAV vector was added at a MOI of 15,000 per well. Twenty-four hours later, the media was replaced with DMEM containing 2% FBS. Forty-eight hours post transduction, the cells were imaged using a Zeiss Axiovert 200 M live-cell imaging microscope (Zeiss, Oberkochen, Germany), and analysed for GFP expression, using flow cytometry on a BD FACS Canto II (BD Biosciences, Franklin Lakes, NJ, USA). To determine the dose response to rAAV, cells were transduced with both HEK293 and baculovirus expression vector (BEV) system produced rAAV at MOIs of 2500, 5000, and 10,000.