Journal: bioRxiv
Article Title: Maintenance of neuronal TDP-43 expression requires axonal lysosome transport
doi: 10.1101/2024.09.30.615241
Figure Lengend Snippet: A) Quantification of BORC KD TDP-43 immunofluorescence microscopy. Both BORC genes tested (S2 and S6) show significant decreases in untagged TDP-43 levels compared to a NT guide, indicated by blue dots. N=4 wells per genotype, 9 images per well (small gray dots). Significant p-values indicated on graph. B) Representative images of TDP-43 immunofluorescence imaging with BORCS6 KD. sgRNA plasmids contain a cytoplasmic BFP, enabling identification of cells expressing the sgRNA. Scale bar represents 20 µm. C) Schematic of pLentiCRISPR plasmid. pLentiCRISPR enables lentiviral delivery of a plasmid expressing an sgRNA of interest under a U6 promoter and Cas9 under an Ef1alpha promoter. This enables knockout of a gene of interest targeted by an sgRNA. D) Representative images of BORCS7 KO TDP-43 IF with lysosomes stained with anti-H4A3 antibody. Scale bar is 20 µm. E) Quantification of TDP-43 immunofluorescence on BORCS7 knockout neurons. Both BORCS7 guides significantly reduced the amount of TDP-43 IF signal compared to a NT guide, light blue dots. N=10 wells, 9 images per well (light gray dots). Significant p-values indicated on graph.
Article Snippet: TDP-43 coding sequence and 3’UTR were cloned into a plasmid expressing 24 SunTag repeats under an Ef1alpha promoter followed by 24 PP7 stem loops (modified from Addgene 74928).
Techniques: Immunofluorescence, Microscopy, Imaging, Expressing, Plasmid Preparation, Knock-Out, Staining
