ha 1 1 000 cat no 3724s cell signaling technology inc  (Cell Signaling Technology Inc)

Journal: Science Advances

Article Title: Self-inactivating AAV-CRISPR at different ages enables sustained amelioration of Huntington’s disease deficits in BAC226Q mice

doi: 10.1126/sciadv.aea8052

Figure Lengend Snippet: ( A ) Schematic representation of targeted human HTT exon 1. sgRNAs HTTg1 and HTTg2 were designed to target the N17 domain–coding DNA sequence, which is located upstream of the CAG repeats. PAM, protospacer-adjacent motif. ( B ) Schematic representation of AAV-SaCas9-HTT sgRNA vector. U6-driven sgRNA and a CMV-driven Cas9 from S. aureus tagged with HA were packaged into a single AAV vector of 4.5 kb. ITR, inverted terminal repeat; NLS, nuclear localization signal sequence; 3×HA, three tandem repeats of the human influenza hemagglutinin (HA) tag. ( C to K ) Validation of human HTT editing in human embryonic kidney (HEK) 293T cells expressing EGFP-hHTT-20Q or EGFP-hHTT-120Q reporter system. (C) SaCas9-HTTg1 and SaCas9-HTTg2 reduced EGFP-fusion protein expression and aggregation in cells, whereas the control SaCas9-sgRNA did not. Scale bar, 100 μm. [(D) and (E)] Quantitative analyses of EGFP-20Q (D) or EGFP-120Q (E) fluorescence intensity. n = 3 or 4; ** P = 0.0083 (20Q), ** P = 0.0087 (120Q), and *** P = 0.0009 (120Q). [(F) to (K)] Western blot detecting EGFP and SaCas9 (anti-HA) protein level in EGFP-hHTT-20Q [(F) to (H)] or EGFP-hHTT-120Q [(I) to (K)] reporter system. [(F) and (I)] SaCas9-HTTg1 and SaCas9-HTTg2 reduced EGFP-hHTT protein expression compared to control SaCas9-sgRNA with equivalent SaCas9 expression level. [(G), (H), (J), and (K)] Quantification of relative EGFP [(G) and (J)] and SaCas9 [(H) and (K)] protein expression. n = 3 per group; ** P = 0.0087 (20Q) and ** P = 0.0069 (120Q). Data were shown as means ± SEM. One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test.

Article Snippet: The following primary antibodies were used: mEM48 (1:1000, mouse; Millipore, MAB5374), S830 (1:5000, sheep, provided by G. Bates from University College London), HA (1:1000, rabbit; Cell Signaling Technology, 3724), HA (1:1000, mouse; Abcam, ab18181), GFAP (1:1000, rabbit; Abcam, ab7260), and NeuN (1:1000, mouse; Abcam, ab104224).

Techniques: Sequencing, Plasmid Preparation, Biomarker Discovery, Expressing, Control, Fluorescence, Western Blot

Journal: Science Advances

Article Title: Self-inactivating AAV-CRISPR at different ages enables sustained amelioration of Huntington’s disease deficits in BAC226Q mice

doi: 10.1126/sciadv.aea8052

Figure Lengend Snippet: ( A ) Experimental timeline in HD mice and corresponding phenotypes at different time points. ( B to I ) Significant reduction of mEM48-positive mHTT aggregates (green) in the striatum from 4-month-injected [(B) to (E)] and 7-month-injected HD mice [(F) to (I)]. Mouse brain samples were collected 4 months postinjection. [(B) and (F)] Representative figures showing that mHTT aggregation was decreased in 4-month-injected HD-SaCas9-HTTg1 (B) and 7-month-injected HD-SaCas9-HTTg1 (F). mHTT (green) and SaCas9 (red) were detected by mEM48 and anti-HA antibody, respectively. Scale bars, 100 μm. Enlarged figures were shown on the right side. Scale bars, 20 μm. [(C) to (E) and (G) to (I)] Quantification of mHTT aggregation signal [(C) and (G)], aggregate number [(D) and (H)], and aggregate size [(E) and (I)] in the striatum in 4-month-injected HD-SaCas9-HTTg1 mice [(C) to (E)] and 7-month-injected HD-SaCas9-HTTg1 mice [(G) to (I)]. n = 3 to 6 per group; *** P < 0.0001 [(C) to (E)], ** P = 0.0031 (D), *** P = 0.0006 [(G), HD-no inj-7M versus HD-SaCas9-7M inj-11M], *** P < 0.0001 [(G), HD-SaCas9-7M inj-11M versus HD-SaCas9-HTTg1-7M inj-11M], *** P = 0.0007 (H), ** P = 0.0082 (I), and * P = 0.0363 (I). Data were shown as means ± SEM. One-way ANOVA with Tukey’s multiple comparisons test. n.s., not significant.

Article Snippet: The following primary antibodies were used: mEM48 (1:1000, mouse; Millipore, MAB5374), S830 (1:5000, sheep, provided by G. Bates from University College London), HA (1:1000, rabbit; Cell Signaling Technology, 3724), HA (1:1000, mouse; Abcam, ab18181), GFAP (1:1000, rabbit; Abcam, ab7260), and NeuN (1:1000, mouse; Abcam, ab104224).

Techniques: Injection