Aav2 helper free system

Recombinant adeno-associated viral (rAAV) vectors were produced using an AAV2 helper-free system (Agilent Technologies), as described in Darcq et al. [46] (link). The rAAV vectors expressed a shRNA sequence under the control of mU6 promoter, along with eGFP under the control of the CMV promoter. The shRNA sequence targeted at the Pdyn transcript (shPdyn, 5′-GGTTGCTTTGGAAGAAGGCTACA-3′) was selected using BLOCK-iT RNAi designer (Life Technologies, Grand Island, NY) and reduced expression of a rat prodynorphin-eGFP fusion in transfected COS-1 cells by 98% compared to a scramble shRNA sequence with no homology to any transcript (shScr, 5′-GCGCTTAGCTGTAGGATTC-3′), as analyzed by fluorescence-activated cell sorting (data not shown).

For primary AAV2 viral vector production, the AAV-2 Helper Free System (Agilent, catalog number: 240071) was used. Briefly, AAV-293 cells were seeded at 70–80% confluency. The cells were then transfected with pAAV-RC, pHelper and pAAV expression plasmid using JetPRIME (Polyplus Transfection, catalog number: 101000046). The cells were harvested after 72 h and subjected to four rounds of freeze/thaw cycles using a dry ice-ethanol bath and a 37 °C water bath. The cells were then centrifuged at 16,000 g for 10 min at room temperature, and the supernatant was transferred to new Eppendorf tubes stored at −80 °C. The physical titers of primary AAV2 viral vectors were determined by qPCR AAV Titer Kit (Applied Biological Materials, Richmond, BC, Canada, catalog number: G931).