Journal: iScience

Article Title: Adeno-associated virus vector system controlling capsid expression improves viral quantity and quality.

doi: 10.1016/j.isci.2023.106487

Figure Lengend Snippet: Figure 4. Novel AAV vector production system is applicable for other serotypes (A) Western blot of replicase (Rep) and capsid (Cap) proteins during adeno-associated virus (AAV) vector production for various serotypes in HEK293 cells. Rep, Cap, and b-actin proteins were detected using anti-Rep, anti-Cap, and anti- b-actin antibodies, respectively. (B–E) AAV vector yield in various serotypes for the Tet-Cap system. Fold difference of AAV vector yield in AAV7 (B), AAV8 (C), AAV9 (D), and AAVrh10 (E) after a change in medium and doxycycline stimulation at 12 h after transfection. Data were normalized with the value of the normal control (RC) samples. (F–I) Western blot of Cap proteins after immunoprecipitation for various serotypes. The same titer of AAV vector (1 3 109– 4 3 109 vg/sample) calculated with qPCR was subjected to immunoprecipitation using ADK8 (AAV7, AAV8, and rh10) and ADK8/9 (AAV9) antibodies, as well as protein A/G magnetic beads, before western blotting. The left-hand figures show representative western blotting images, and the right-hand figures the fold difference of band intensities for VP1, VP2, and VP3 proteins between RC and Tet-Cap samples in each panel. These panels indicate the data for AAV7 (F), AAV8 (G), AAV9 (H), and AAVrh10 (I), respectively. Experiments were independently performed at least three times for statistical analysis. Asterisks in each panel indicate the following: * = p< 0.05, ** = p< 0.01. Error bars indicate the standard error of the mean.

Article Snippet: AAV serotypes 1, 7, 8, 9, and rh10 capsid (Cap) plasmids (pAAV2/1, pAAV2/7, pAAV2/8, pAAV2/9n, and pAAV2/rh10) were gifted by James M. Wilson (Addgene plasmids: 112862, 112863, 112864, 112865, and 112866).

Techniques: Plasmid Preparation, Western Blot, Virus, Transfection, Control, Immunoprecipitation, Magnetic Beads