β actin  (Proteintech)

Journal: Synthetic and Systems Biotechnology

Article Title: Enhanced AAV production via rational design of a novel pHelper vector integrated with HSV-1 helper genes

doi: 10.1016/j.synbio.2025.12.018

Figure Lengend Snippet: Evaluation of viral helper factors to improve AAV yield via four-plasmid transfection . (A) Schematic diagram of AAV vector production via four-plasmid transfection system. Equimolar ratio of pHelper, pAAV-RC, pAAV-EGFP and viral helper factor-pcDNA3.1 (pcDNA3.1 carrying helper virus factors) were co-transfected into HEK293 cells for AAV5 production. (B) AAV5 vector yield in HEK293 cells via four-plasmid transfection. VG titers in cell suspensions were measured at 72 hpt using EGFP-specific qPCR. Data were calculated from three biological replicates. Statistical was analyzed by one-way ANOVA, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (C) Total replicated viral DNA measured at 72 hpt. (D–E) Relative transcript levels of Cap and Rep measured at 24 hpt using GAPDH as the reference gene. Transcript levels are presented as fold-change relative to the control group. Data were calculated from three biological replicates. One-way ANOVA analysis indicates a statistically significant difference between control group with various helper virus factors group (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001). (F) Western blot analysis of AAV Rep and Cap protein expression at 72 hpt using four-plasmid transfection. β-actin was detected as a loading control.

Article Snippet: The blotted membranes were probed with anti-Rep antibody (1:500, Progen, Germany), anti-AAV VP1/VP2/VP3 monoclonal antibody (1:2000, Progen, Germany) and anti-β-actin antibody (1:1000, Proteintech, China).

Techniques: Plasmid Preparation, Transfection, Virus, Control, Western Blot, Expressing

Journal: Synthetic and Systems Biotechnology

Article Title: Enhanced AAV production via rational design of a novel pHelper vector integrated with HSV-1 helper genes

doi: 10.1016/j.synbio.2025.12.018

Figure Lengend Snippet: Integration of viral helper factors into pHelper for AAV production . (A) Schematic diagram of the engineered pHelper constructs, in which single or dual helper virus genes (UL5, UL12, ICP8, and ICP22) were inserted between the VA RNA and E4 regions. (B) AAV5 vector yield in HEK293 cells co-transfected with pAAV-EGFP, pAAV-RC5, and pHelper or constructed helper plasmid. VG titers in cell suspensions were measured at 72 hpt using EGFP-specific qPCR. Data were calculated from three biological replicates. Statistical was analyzed by one-way ANOVA, ∗∗ p < 0.01. (C) Total replicated viral DNA measured at 72 hpt. and relative transcript levels of Cap (D–E) Relative transcript levels of Cap and Rep measured at 24 hpt using GAPDH as the reference gene. Relative transcript is presented as fold-change to pHelper group. Data were calculated from three biological replicates. One-way ANOVA analysis indicates a statistically significant difference between control group with various helper virus factors group (∗ p < 0.05). (F) Western blot analysis of AAV Rep and Cap protein expression at 72 hpt co-transfected with pAAV-EGFP, pAAV-RC5, and pHelper, UL12-pHelper or UL12-ICP22-Helper. β-actin was detected as a loading control. (G) Assessment of helper plasmid uptake per mL co-transfected with pAAV-EGFP, pAAV-RC5, and pHelper or constructed helper plasmid. Statistical significance was calculated by one-way ANOVA with Tukey's multiple comparisons, ∗ p < 0.05.

Article Snippet: The blotted membranes were probed with anti-Rep antibody (1:500, Progen, Germany), anti-AAV VP1/VP2/VP3 monoclonal antibody (1:2000, Progen, Germany) and anti-β-actin antibody (1:1000, Proteintech, China).

Techniques: Construct, Virus, Plasmid Preparation, Transfection, Control, Western Blot, Expressing

Journal: Synthetic and Systems Biotechnology

Article Title: Enhanced AAV production via rational design of a novel pHelper vector integrated with HSV-1 helper genes

doi: 10.1016/j.synbio.2025.12.018

Figure Lengend Snippet: A mini-pHelper containing deletions within the E4 and E2a genes maintained AAV productivity . (A) Schematic diagram of the mini-pHelper plasmid construction by deleting 0.9 kb of the E2a gene and 2.5 kb of the E4 gene. Black arrows indicate the direction and length of protein coding sequence in the plasmid. (B) AAV5 vector yield in HEK293 cells co-transfected with pAAV-EGFP, pAAV-RC5, and pHelper, pHelper-E2aΔ0.9 kb or mini-pHelper. VG titers in cell suspensions were measured at 72 hpt using EGFP-specific qPCR. Data were calculated from three biological replicates. Statistical was analyzed by one-way ANOVA, ∗ p < 0.05. Relative transcript levels of Rep (C) and Cap (D) using GAPDH as the reference transcript. Relative transcript is presented as fold-change to pHelper group. Data were calculated from three biological replicates. Statistical was analyzed by one-way ANOVA, ∗ p < 0.05. (E) Western blot analysis of AAV Rep and Cap in HEK293 cells co-transfected with pAAV-EGFP, pAAV-RC5, and pHelper, pHelper-E2aΔ0.9 kb or mini-pHelper. β-actin was detected as a loading control.

Article Snippet: The blotted membranes were probed with anti-Rep antibody (1:500, Progen, Germany), anti-AAV VP1/VP2/VP3 monoclonal antibody (1:2000, Progen, Germany) and anti-β-actin antibody (1:1000, Proteintech, China).

Techniques: Plasmid Preparation, Sequencing, Transfection, Western Blot, Control

Journal: Synthetic and Systems Biotechnology

Article Title: Enhanced AAV production via rational design of a novel pHelper vector integrated with HSV-1 helper genes

doi: 10.1016/j.synbio.2025.12.018

Figure Lengend Snippet: Integration of UL12 and ICP22 genes into the mini-pHelper further enhanced AAV vector production . (A) Schematic diagram of the engineered various constructed helper plasmid. (B) AAV5 vector yield in HEK293 cells co-transfected with pAAV-EGFP, pAAV-RC5, and constructed helper plasmid. VG titers in cell suspensions were measured at 72 hpt using EGFP-specific qPCR. Data were calculated from three biological replicates. Statistical was analyzed by one-way ANOVA, ∗ p < 0.05. ∗∗ p < 0.01. (C) Total replicated viral DNA measured at 72 hpt. (D–E) Relative transcript levels of Cap and Rep measured at 24 hpt using GAPDH as the reference gene. Relative transcript is presented as fold-change to pHelper group. Data were calculated from three biological replicates. Statistical was analyzed by one-way ANOVA, ∗ p < 0.05, ∗∗ p < 0.01. (F) Western blot analysis of AAV Rep and Cap protein expression co-transfected with pAAV-EGFP, pAAV-RC5, and constructed helper plasmid. β-actin was detected as a loading control. (G) Proportion of encapsulated genomes for AAV5 in HEK293 cells co-transfected with pAAV-EGFP, pAAV-RC5, and constructed helper plasmid. Analysis of empty to full AAV particles using anion-exchange chromatography methods on a 0.1 mL CIMmultus monolith and expressed as a fold-change compared to the pAAV-RC. Statistical was analyzed by one-way ANOVA, ∗ p < 0.05.

Article Snippet: The blotted membranes were probed with anti-Rep antibody (1:500, Progen, Germany), anti-AAV VP1/VP2/VP3 monoclonal antibody (1:2000, Progen, Germany) and anti-β-actin antibody (1:1000, Proteintech, China).

Techniques: Plasmid Preparation, Construct, Transfection, Western Blot, Expressing, Control, Chromatography

Journal: Synthetic and Systems Biotechnology

Article Title: Enhanced AAV production via rational design of a novel pHelper vector integrated with HSV-1 helper genes

doi: 10.1016/j.synbio.2025.12.018

Figure Lengend Snippet: UL12-ICP22-miniHelper improves productivity for various capsid serotypes . Vector yields and Western blot analysis of AAV Rep and Cap expression for AAV2/1 (A), AAV2 (B), AAV2/5 (C), AAV2/6 (D), AAV2/9 (E). HEK293 cells were co-transfected with pAAV-EGFP, pRC, and pHelper or UL12-ICP22-miniHelper. VG titers in cell suspensions were measured at 72 hpt using EGFP-specific qPCR. Data were calculated from three biological replicates. Statistical was analyzed by one-way ANOVA, ∗ p < 0.05. ∗∗ p < 0.01. β-actin was detected as a loading control. (F) Summary of AAV vector titers across multiple serotypes using either the pHelper or the UL12-ICP22-miniHelper plasmid. Fold increases represent the ratio of VG titers obtained with UL12-ICP22-miniHelper to those obtained with pHelper.

Article Snippet: The blotted membranes were probed with anti-Rep antibody (1:500, Progen, Germany), anti-AAV VP1/VP2/VP3 monoclonal antibody (1:2000, Progen, Germany) and anti-β-actin antibody (1:1000, Proteintech, China).

Techniques: Plasmid Preparation, Western Blot, Expressing, Transfection, Control