Recombinant AAV2/5 for Astrocyte-Specific Expression

Recombinant AAV2/5 was prepared by using AAV Helper-Free System (Agilent Technology) with some modification. To generate an AAV2/5 capable of expressing mCherry-PKI fusion protein in astrocytes, AAV2/5 rep, and cap plasmid was purchased from Penn Vector Core (University of Pennsylvania) and pZac2.1 gfaABC1D-tdTomato^{61 (link)} was a gift from Baljit Khakh (Addgene plasmid # 44332). To construct pZac2.1 gfaABC1D-mCherry–PKI, the cDNA for mCherry-PKI was amplified from p4mt-mCherry-PKI that was kindly provided by Dr. Giulietta Di Benedetto^{62 (link)} by using following primers; Forward 5’-AAGCTTGAATTCGCCACCATGGTGAGCAAGGGCGAGGAGGA-3’ and reverse 5’-AAGCTTGCGGCCGCCTATGACTCGGACTTAGCAG-3’. The tdTomato portion of pZac2.1 gfaABC1D-tdTomato was replaced with mCherry-PKI at EcoR I and Not I site. After DNA sequence validation, these plasmids were used for AAV2/5 production. Viruses were prepared according to manufacturer’s instruction (AAV Helper-Free System; Agilent Technology) and purified by a column chromatography using a ViraTrap AAV purification kit (Biomiga) as described in the manufacturer’s protocol. Viral titers were determined using a quantitative real-time PCR method. The both AAV2/5 titers were 2.0 × 10¹² GC/ml and transduction was performed to ONH astrocytes with 400,000 MOI.