Generation of Viral Vectors for Selective Expression

Adeno-associated virus (AAV) was generated using AAV-DJ Helper Free system (Cell Biolabs) as we published previously.^{9 (link)} To express ExSYTE selectively in the ‘Subthreshold fear conditioning’ neuronal population, the ExSYTE cassette was located in a reverse direction with two lox2272 and loxP loci (AAV-FLEX-ExSYTE) as illustrated in Figure 4D. The ExSYTE or ExSYTE^H64A fused with the P2A peptide-EGFP was cloned into the modified pAAV-MCS and transfected with pAAV-DJ and pHelper into 293AAV cells (Cell Biolabs). Transfected cells were lysed, and AAVs were purified with HiTrap heparin columns (GE Health) as previously described.^{69 (link)} AAV-EF1α-mCherry-IRES-Cre was purchased from Addgene (Cat. #55632-AAV8).