Paav mcs

To produce adeno-associated virus (AAV) vectors, ND1-P2A-mCherry was inserted into pAAV-FLEX-GFP vector (Addgene) to replace GFP to generate pAAV-Flex-CAG-ND1-P2A- mCherry. mCherry was inserted into pAAV-FLEX-GFP to replace GFP and was used as a negative control. Human GFAP promoter (1.6 kb) was inserted into pAAV-MCS (Cell Biolabs) to replace CMV promoter, then Cre was subcloned into pAAV-MCS to generate pAAV-hGFAP-Cre. The backbone of pAAV-GFAP::ND1-GFP and pAAV-GFAP::GFP vectors is pAAV-MCS (Cell Biolabs, Inc.). CMV promoter was changed to GFAP promoter, and GFP or NeuroD1-GFP was inserted to MCS. Recombinant AAV9 was produced by HEK293T cell line. The purification method used Iodixanol Gradient Solutions (D1556, Optiprep, Sigma) and desalting and concentrating on Merck Amicon Ultra-15 Centrifugal Filters (UFC910008, Millipore). Purified AAV viruses were titered using QuickTiter AAV Quantitation Kit (VPK-145, Cell biolabs).

Previously characterized CII T-cell epitopes were synthesized in a sequence derived from the native murine collagen II (Col2A1) gene cDNA sequence by Genescript (Piscataway, NJ). The resulting construct was cloned via Gibson assembly (New England Biolabs, Rowley, MA) into either pAAV-MCS or pacAd5 CMV K-N (all from Cell Biolabs, San Diego, CA) to generate the pAAV-MCS: CII or pacAd5 CMV K-N: CII constructs.

Recombinant AAV vectors that expressed, under the influence of the Myh11 promoter, either the GRK2 or the GFP that served as the infection control were generated. We selected AAV-DJ8 as a virus serotype in this study after the vasculature expression trial of GFP with Myh11 promotor. The Grk2 gene and Myh11 promoter were cloned from a wildtype C57BL/6 J mouse that was purchased from Clea Japan (Tokyo, Japan). The GFP-expressing plasmid was procured from Cell Biolabs (AAV-400, San Diego, California, United States). For pAAV-DJ8-GRK2 or pAAV-DJ8-GFP plasmid construction, pAAV-MCS were purchased from Cell Biolabs (VPK-410). After inserting Grk2 or GFP and exchanging CMV promotor to Myh11 promotor in pAAV-MCS, it was transfected into human embryonic kidney 293 cells together with pAAV-DJ8 and pHelper purchased from Cell Biolabs (VPK-420-DJ-8, 3402-02) to produce AAV-DJ8-GRK2 or AAV-DJ8-GFP vector. The vector was then purified, and concentrated by two cycles through the Amicon Ultra-15 centrifugal filters (UFC910024; Merck Millipore, Burlington, Massachusetts, United States). The purified viral preparations were stored in phosphate-buffered saline, and physical particle titers were determined using competitive PCR. The primers used for cloning Grk2 gene and Myh11 promoter are listed in S1 Materials and Methods.