Generating Plasmids for Marmoset D1R and D2R Knockdown

AAV vectors were based on the AAV helper-free system (Agilent Technologies, CA, USA). To generate plasmids encoding shRNA driven by a U6 promoter, a 0.7-kb PvuII fragment from pSilencer 2.1-U6 NEO (Ambion Life Technologies, MA, USA) was inserted into the blunted MluI site of pAAV-hrGFP (Agilent Technologies)52 (link). The CMV promoter for hrGFP was replaced by the human synapsin I promoter. A shRNA cassette was inserted into the plasmid digested with BamHI and HindIII located directly below the U6 promoter52 (link). The shRNA target sequence for the marmoset D1R was 5′-GTCGAATGTTCTCAACCAGAA-3′, and that for the marmoset D2R was 5′-GGACAGACCTCACTACAATTA-3′. Silencing efficacy was evaluated using the Dual-Luciferase reporter assay system (Promega, WI, USA)53 (link). The shRNAs with more than 80% knockdown efficacy in vitro were identified (see Supplementary Fig. 1), and those having high efficacy were tested more than twice.