Paav mcs vector

Murine AIBP was fused with fibronectin secretion sequence (FIB) at the N terminus and 6×His at the C terminus (FIB-AIBP-His). FIB-AIBP-His was cloned into pAAV-MCS vector (Agilent Technologies). AAV-293 cells (Agilent Technologies) were transfected with 20 μg each of pAAV-FIB-mAIBP-His, pAAV-DJ/8 (Cell Biolabs), and pHelper DNA (Cell Biolabs). Subsequent steps of virus harvest, purification, and storage were performed according to published protocols (81 (link)). Viral DNA was extracted from purified virus, and the number of gene copies (gc) was determined using quantitative PCR (qPCR) with primers for the inverted terminal repeats (TaKaRa Bio Inc.).

We used AAV serotype 9 for our experiments because of its high expression level and its brain tropism.^{54 (link)} The fragments corresponding to 2xFLAG-α-globin and β-globin-MYC were cloned into the pAAV-MCS vector (Agilent Technologies, Santa Clara, CA, USA), which was used to produce recombinant AAV vectors. In addition, we use pAAV-MCS vector as control. AAV of serotype 9 were generated in HEK 293 T cells, using a triple-plasmid co-transfection for packaging. Viral stocks were obtained by CsCl₂ gradient centrifugation. Titration of AAV viral particles was performed by real-time PCR quantification of the number of viral genomes. The viral preparations had the following titres: AAV9-2xFLAG-α-globin 7 × 10¹³ viral genome particles/ml, AAV9-β-globin-MYC 8.8 × 10¹³ viral genome particles/ml and AAV9-control 7.5 × 10¹² viral genome particles/ml.