aaaag ctttt  (ATCC)

Journal: Science Advances

Article Title: AAGGG repeat expansions trigger RFC1 -independent synaptic dysregulation in human CANVAS neurons

doi: 10.1126/sciadv.adn2321

Figure Lengend Snippet: ( A ) Schematic of brain, central and peripheral nervous system regions affected in CANVAS (left), and potential mechanisms of repeat toxicity in CANVAS (right). ( B ) Repeat architecture of the expanded locus and CRISPR gRNA design to remove the AAGGG/CCCTT repeat expansion by nonhomologous end joining (NHEJ). ( C ) Endpoint PCR of gDNA extracted from CANVAS patient– and control-derived iPSC lines and CANVAS and control cerebellum tissue utilizing the primer pair outlined in (B) to screen for the presence of WT repeat, mutant repeat expansion, or deletion of expanded repeat. ( D ) Chromatogram of Sanger sequencing identifying AAGGG/CCCTT allele deletion in heterozygous isogenic line indicating the expected NHEJ join point compared to the control iPSC line. ( E ) Schematic outlining the repeat copy number per allele for each of the patient-derived iPSC lines used as identified by Oxford Nanopore gDNA targeted long-read sequencing. Green, sub-pathogenic repeat length; Red, pathogenic repeat length. Error bars indicate confidence in exact copy number calls. The “*” indicates lower boundary for repeat copy number from the longest read observed due to the absence of reads spanning the full repeat.

Article Snippet: Probes against the AAAAG, AAGGG, CTTTT, and CCCTT sequences (table S2) were purchased from molecularinstruments.org and applied according to the manufacturer’s protocol.

Techniques: CRISPR, Control, Derivative Assay, Mutagenesis, Sequencing

Journal: Science Advances

Article Title: AAGGG repeat expansions trigger RFC1 -independent synaptic dysregulation in human CANVAS neurons

doi: 10.1126/sciadv.adn2321

Figure Lengend Snippet: ( A ) Schematic of potential peptide products from sense and antisense strand of the repeat expansion locus. ( B ) Quantification of foci positive neurons for control ( n = 3) and CANVAS ( n = 3) patient iPSC-derived neurons (1100 to 2000 cells per group per probe). n = 2 biological replicates from three independent patient-derived cell lines. Representative confocal images are shown in fig. S2B. ( C ) Immunoblot from HEK293 cells expressing plasmids encoding intronic sense or antisense AAGGG/CCCTT repeat reporters in the +0/+1/+2 reading frames (left) and Nano-luciferase expression assay quantification (right). n = 7 biological replicates. Data were analyzed by one-way ANOVA with Sidak’s post hoc multiple comparison tests. ( D ) ICC of HEK293 cells transfected with plasmids encoding intronic sense or antisense AAGGG/CCCTT repeat reporters with C-terminal triple tags in the +0/+1/+2 reading frames. ( E ) Expression analysis of lysates from HEK293 cells transfected with control plasmid +2 Sense (AAGGG) 61 plasmid using anti-FLAG M2 (1:1000) and anti-KGREG (1:100) antibodies (left). ( F ) Left: IHC of control and RFC1 expansion CANVAS patient postmortem cerebellar vermis tissue stained with sense anti-KGREG antibody (1:100, acid AR). Scale bars, 500 μm (4×), 50 μm (60×), and 20 μm (inset). Right: Rater blinded quantification of all 20 postmortem tissues (tissue images and quantification for each sample in fig. S4). ( G ) Cumulative hazard plot for rat cortical neurons expressing CGG100 (positive control) or CANVAS intronic expression plasmids containing 61 repeats of the indicated type over 10 days. Results from eight technical replicates/three biological replicates; n = numbers of cells assessed per condition. ns, not significant. *hazard ratio = 1.339, P = 0.025, Cox proportional hazards analysis.

Article Snippet: Probes against the AAAAG, AAGGG, CTTTT, and CCCTT sequences (table S2) were purchased from molecularinstruments.org and applied according to the manufacturer’s protocol.

Techniques: Control, Derivative Assay, Western Blot, Expressing, Luciferase, Comparison, Transfection, Plasmid Preparation, Staining, Positive Control