Figure 6.

Difference in the Rep78/Rep52 expression ratio improves AAV vector quantity and quality

(A) Diagram of various replicase (Rep) constructs. CMV = CMV promoter; Rep2 = full length AAV2 Rep gene; 78 = Rep78 gene; 52 = Rep52 gene; IRES = internal ribosome entry site sequence; P2A = 2A sequence of porcine teschovirus-1.

(B) Western blot of Rep proteins using various Rep constructs in HEK293 cells. Rep and β-actin proteins were detected using anti-Rep and anti-β-actin antibodies, respectively. The white triangle shows Rep52-P2A-Rep78 fusion proteins.

(C) Western blot of capsid (Cap) proteins during adeno-associated virus (AAV) vector production using various Rep constructs in HEK293 cells. Cap and β-actin proteins were detected using anti-Cap and anti-β-actin antibodies, respectively.

(D) AAV vector yields for various Rep expressions. Fold difference in AAV vector yields between the normal control (RC2) and various Rep-transfected groups after a change in medium and doxycycline stimulation at 12 h after transfection.

(E) Fold differences in qPCR/ELISA corresponding with the full/empty particle ratio during various Rep expressions. Data were normalized with the value of the RC2 sample to calculate fold difference.

(F) Infectivity data for AAV vectors produced using various Rep constructs. The 2v6.11 cells were infected with AAV2 (500 vg/cell) and observed at 96 h after infection. These data indicate infectivity of the AAV vector (EGFP; green) in AAV2 produced using various Rep transfections. The blue signal shows nuclei (Hoechst 33342). White bar shows 100 μm. 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.