AAV-293 cells (Agilent Technologies) were transfected with AAV vectors (pAAV-LSL-GFP-shRNAmirs) and helper vectors (pAd helper vector and pAAV2/5 packaging vector) following a standard CaCl2 method with some modifications (61). Transfected cells were harvested 48 hours later and lysed by three rounds of freeze-thaw cycles. The lysates were further sonicated, and virus-containing fractions were precipitated by adding ammonium sulfate. Then, the viruses were enriched by OptiPrep density gradient ultracentrifugation at 60,000 rpm for 1.5 hours, followed by concentration of viruses using Amicon Ultra Centrifugal filter unit (100,000 Da molecular weight cut-off) (Millipore). Virus titers were estimated using a qPCR-based method to detect inverted terminal repeat (ITR) sequence as described previously (50). The following primers and probe were used: AAV2 ITR F, 5′-GGAACCCCTAGTGATGGAGTT-3′; AAV2 ITR R, 5′-CGGCCTCAGTGAGCGA-3′; and AAV2 ITR probe, 5′-CACTCCCTCTCTGCGCGCTCG-3′.

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