Paav dj8

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.

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.).