retrograde aav helper vector  (Addgene inc)

Journal: ACS Synthetic Biology

Article Title: Enhanced Epigenetic Modulation via mRNA-Encapsulated Lipid Nanoparticles Enables Targeted Anti-inflammatory Control

doi: 10.1021/acssynbio.5c00188

Figure Lengend Snippet: Co-delivery of Myd88 repressor ZFR11 and gene therapy related antigen modulates humoral immunity in AAV preimmunized mice. (A) Schematic representation of experimental design. C57BL/6J mice received two retro-orbital injections of AAV2-GFP (5 × 10 11 GC) 2 weeks apart. Two weeks later, mice were administered weekly doses of LNP-encapsulated mRNA (0.7 mg kg –1 ) encoding either ZFR11-VP1-mCherry ( N = 10), VP1 only ( N = 10), or no LNP/PBS control ( N = 6) for 4 weeks. Two weeks after the final vaccination, mice received a single intravenous coinjection of AAV2-mCherry (5 × 10 11 GC) and ZFR11-LNP (0.7 mg kg –1 ). Analyses were performed 3 weeks post-AAV2 challenge. (B) qRT-PCR analysis of mCherry expression in liver and lung tissues ( N = 8–10 mice per treatment group, N = 6 for PBS). Data are presented as mean ± SEM Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test. * P ≤ 0.05 was considered statistically significant (C) Left: Representative immunohistochemical staining for mCherry in liver sections. Right: Quantification of mCherry staining intensity (ZFR11-VP1-mCherry-306O 10 : N = 9, PBS: N = 5, staining negative control/mCherry uninjected: N = 2). Data are presented as mean ± SEM Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test. * P ≤ 0.05 was considered statistically significant. (D) ELISA measurement of plasma anti-AAV2 total IgG and IgG2b levels. Results shown as fold change in optical density at day 35 (2 weeks postvaccination) relative to day 0 (prevaccination baseline) ( N = 9–10 per treatment group, N = 6 for PBS). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test. * P ≤ 0.05 was considered significant. (E) ELISA measurement of serum anti-AAV2 neutralizing antibody levels showing a 32% reduction in the ZFR11-VP1-mCherry group compared to VP1 control at day 35 (2 weeks postvaccination) relative to day 0 (prevaccination baseline) ( N = 9–10 mice per treatment group, N = 6 mice for PBS). Statistical analysis was performed using the unpaired parametric t test. * P ≤ 0.05 was considered significant. (F) Time-point analysis of plasma total IgG, IgG2b, and neutralizing antibodies titers in Relative Light Units (RLU) at day 0 (prevaccination baseline), day 35 (2 weeks postvaccination), and day 45 (10 days after AAV2-mCherry rechallenge) ( N = 9–10 per treatment group).

Article Snippet: The mCherry construct was a premade AAV vector purchased from PackGene Biotech, (ssAAV-CAG-mCherry.WPRE.SV40pA, cat. no. AAV-EA022).

Techniques: Control, Quantitative RT-PCR, Expressing, Immunohistochemical staining, Staining, Negative Control, Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: eLife

Article Title: Expansion-assisted selective plane illumination microscopy for nanoscale imaging of centimeter-scale tissues

doi: 10.7554/elife.91979

Figure Lengend Snippet: Figure 5. Expansion and imaging of a large volume of macaque brain. A 1 cm×1 cm×1.5 cm block of macaque primary motor cortex was expanded (3×) and imaged on the ExA-SPIM (Videos 7–9). Corticospinal neurons were transduced by injecting tdTomato-expressing retro-AAV into the spinal cord. (a–b) Maximum intensity projections of the imaged volume pseudo-colored by depth. The axes descriptors in (a) indicate the 5×3 tiling used to image this volume. (c–f) Fine axonal and dendritic structures including descending axons, collaterals, and dendritic spines are clearly discernible in the images throughout the entire volume. See Video 10. Images are displayed as maximum intensity projections with the following thicknesses: (a) 23 mm, (b) 45 mm, (c–f) 1 mm. Inset scale bars correspond to 10 µm post tissue expansion unless otherwise specified.

Article Snippet: Spinal injection procedure and motor cortex collection To retrogradely label corticospinal neurons in the hand- wrist and trunk regions of primary motor cortex in a pigtail macaque, we injected a retro AAV vector (rAAV2- CAG- tdTomato; Addgene plasmid #59462 packaged in- house, titer of 1.88×1013) into the left lateral funiculus and ventrolateral part of the gray matter in the C6/C7 spinal segments.

Techniques: Imaging, Blocking Assay, Expressing