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. 2022 Jun 8;13:3180. doi: 10.1038/s41467-022-30778-7

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Muscle-specific Crk overexpression by the HSA-promoter. 16% of all obtained animals were transgene-positive tested (n = 51 WT/10 Crk-tg [E18.5]). b, c Increased CRK levels in Crk-tg quadriceps muscles. RALA served as loading control (Western blot, n = 6 WT/9 Crk-tg, WT = littermates, Mann-Whitney-U test, two-tailed, ***p = 0.0004 [E18.5]). d Co-transfection of SP-dCas9-VPR64 vector and sgRNA vectors for Crk activation in C2 cells. Selected sgRNA C (green) for further analysis (TaqMan assay, control = transfections of unrelated sgRNAs n = 15, sgRNAs A-E tested in independent transfections n = 3, Mann-Whitney-U test, one-tailed, **p = 0.0049). Gapdh served as control. e Pax7-Cre-driven dCas9-SPH³³ activation system with selected Crk-sgRNA. f Pax7-Cre-dependent dCas9-SPH expression in quadriceps muscles of Pax7-Cre^+/−//CAG-LSL-dCas9-SPH^+/− mice (Western blot, n = 3 control/3 Crk-SPH, control = sgRNA^+/+ [E18.5]). RALA served as control. g Increased expression of Crk in quadriceps muscle using the Pax7-Cre-dependent dCas9-SPH system in combination with Crk-sgRNA expression compared to control (TaqMan assay, n = 4 control/3 Crk-SPH, control = sgRNA^+/+, Mann-Whitney-U test, one-tailed, *p = 0.0286 [E18.5]). Gapdh served as control. h Whole-mount bungarotoxin (BTX) staining of hemi-diaphragms of Crk overexpressing Crk-tg (n = 9) and Crk-SPH (n = 3) animals compared to WT (scale-bar: 600 µm/100 µm [E18.5]). i Mean AChR cluster size in paraspinal muscles on transversal sections of WT (n = 3) and Crk-tg (n = 3) animals (WT = littermates, Mann-Whitney-U test, one-tailed, *p = 0.05 [E18.5]). j Body weight of control (n = 5) and Crk-SPH (n = 6) pups (control = sgRNA^+/+ littermates, Mann-Whitney-U test, one-tailed, ns = not significant [E18.5]). k Morphology of control and Crk-SPH pups; Crk-SPH animals appeared cyanotic (control = sgRNA^+/+ littermates, scale-bar: 1 cm [E18.5]). l Percentages of Crk-SPH animals at postnatal day 21 (P21) correspond to expected Mendelian ratios (likelihood 12.5%). Crk-SPH genotype (sgRNA^+/−//dCas9^+/−//Pax7-Cre^+/−) is absent at P21 (n = 38 control/0 Crk-SPH; control = littermates). m, n GSEA of control (n = 3) vs. Crk-tg (n = 3) quadriceps muscles transcriptome data (WT = littermates, Nominal p-value (NOM p-val), p = 0.000 (m, n) [E18.5]). Source data are provided as a Source Data file. Data are presented as mean values +/− SEM in c, d, g and I, j.