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Fig. 2 | Acta Neuropathologica Communications

Fig. 2

From: Distinct functional consequences of ECEL1/DINE missense mutations in the pathogenesis of congenital contracture disorders

Fig. 2

Generation of DINE G607S knock-in mice using CRISPR/Cas9. a The original sequence in the DINE locus was changed into a knock-in allele containing a pathogenic missense mutation (G607S). Four exons (Ex12–15) are shown in black boxes. The sgRNA target sequence and PAM sequence are marked with an underline and a square, respectively. The mutated nucleotide is shown in orange in the knock-in allele. The amino acid sequence of the original DINE protein and that of the mutated protein are juxtaposed with the base sequences in the wild-type allele and the knock-in allele, respectively. b Electropherograms showing DNA sequencing results from two G607S founder mice (Founder 1 and Founder 2). The intact peak (g) and mutated peak (a) can be observed at the mutated site (shown by the black arrow head). c Off-target analyses with the mismatch-specific endonuclease were performed on five potential sites (OT1-OT5) using PCR products from the founder tail genomic DNA. Cleaved bands were specifically detected at the on-target site in the founder mice, whereas no cleaved bands were detected on any potential off-target sites. dk Motor nerves were visualized by GFP immunoreactivity to compare the innervation pattern between E17.5 wild-type and homozygous mutant hindlimb muscles. The number of motor nerve terminals was significantly reduced in G607S mutant gracilis anterior (dg) and rectus femoris (hk) muscles. Mean ± SEM, two-tailed Student’s t test, **p < 0.01, n = 5. Scale bar: 100 μm (df, hj)

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