Journal: iScience

Article Title: Downregulation of UBE4B promotes CNS axon regrowth and functional recovery after stroke

doi: 10.1016/j.isci.2022.105885

Figure Lengend Snippet:

Article Snippet: pAAV-U6-gRNA (for HAUSP; 5 gRNA pools)-CAGminin-GFP , Vigene Biotechnologies , .

Techniques: Recombinant, Software

Journal: Investigative Ophthalmology & Visual Science

Article Title: AAV-CRISPR/Cas9–Mediated Depletion of VEGFR2 Blocks Angiogenesis In Vitro

doi: 10.1167/iovs.17-21902

Figure Lengend Snippet: Establishment of dual AAV vectors for delivering CRISPR/Cas9 and confirmation of pICAM2 specificity in vascular ECs. (A) Schematic of AAV-U6-sgRNA-pICAM2-GFP. The hSyn promoter in AAV-SpGuide was substituted by pICAM2 by XbaI/SalI. A protospacer sequence could be cloned into this vector by SapI for synthesizing sgRNA. (B) Schematic of AAV-pICAM2-SpCas9. The ICAM2 promoter was used to replace the Mecp2 promoter in the AAV-SpCas9 by using Xbal/AgeI for driving expression of SpCas9 specifically in vascular ECs. (C–E) ARPE-19 cells and HRECs were grown to 50% confluence in 48-well plates, AAV5-pICAM2-GFP, AAV5-pICAM2-SpCas9, or AAV5-CMV-GFP (3.75 × 10 12 GC/mL) were added into the wells (2 μL/well). Two days later the cells were photographed by fluorescence microscopy (C, E), and the lysates of the cells transduced by AAV5-pICAM2-SpCas9 were subjected to Western blot analysis with antibodies against Cas9 and β-actin (E). These experiments were repeated at least three times.

Article Snippet: The pAAV-U6-sgRNA-pICAM2-GFP vector originated from AAV-SpGuide (Cat. 60958; Addgene) by replacing the hSyn with the PCR-amplified promoter of intercellular adhesion molecule 2 (ICAM2) from genomic DNA of HRECs, using Xba l/ Sal I as described previously.

Techniques: CRISPR, Sequencing, Clone Assay, Plasmid Preparation, Expressing, Fluorescence, Microscopy, Western Blot

Journal: Annals of Neurology

Article Title: Imaging Net Retrograde Axonal Transport In Vivo: A Physiological Biomarker

doi: 10.1002/ana.26329

Figure Lengend Snippet: CRISPR Cas9 Oligonucleotide Sequences

Article Snippet: Backbone AAV‐sgRNA , Addgene 60958 , pAAV‐U6sgRNA_hSyn‐GFP‐KASH‐bGH.

Techniques: CRISPR, Sequencing, Plasmid Preparation