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We previously generated a DINE knock-in mouse line carrying C760R as a relevant model of ECEL1-mutated DA, and detected axonal arborization defects of motor nerves in homozygous C760R mutant limb muscles [24]. However, given that the observed expressivities vary in some affected regions in patients with ECEL1 mutations [2, 30], phenotypic comparison with another knock-in mouse with a distinct pathogenic mutation is necessitated to judge whether axonal arborization defects are a common mechanism in the pathogenesis of ECEL1-mutated DA. Notably, Shaaban et al. have reported two siblings with a missense c.1819G > A mutation (p.G607S) (Fig. 1) in the ECEL1 gene that presented with significant ophthalmoplegia and less pronounced contractures in the distal joints of lower limbs [30]. Because the symptoms did not meet the major criteria for the diagnosis of DA, the authors concluded that the two siblings differed from other patients with different ECEL1 pathogenic mutations. To experimentally compare the pathogenic effects between the C760R and G607S mutations, we have generated a DINE knock-in mouse line carrying G607S using the CRISPR/Cas9 system. We designed a target sequence of sgRNA in the region close to the mutation site, as well as a 90 bp single-stranded DNA (ssDNA) with the pathogenic mutation as the DNA template (Fig. 2a). The CRISPR/Cas9 tools were injected into 200 mouse zygotes and then 158 normally developed two-cell embryos were transferred into recipient female mice. A total of 71 mice were born normally. We performed sequencing analyses using the PCR amplified target region to verify the genotype of the CRISPR-injected mice and successfully obtained 7 F0 mutant mice. We selected two male mice (Founder 1 and Founder 2) with a dominant mutated peak in electropherograms (Fig. 2b) and used these founder mice for expansion of the mouse colony. Off-target analyses using a mismatch cleavage enzyme showed no off-target mutations at five potential sites in the founders (Fig. 2c). The results were also confirmed using direct sequencing analyses (data not shown). After confirmation of the transmission of the desired mutation from the founders to their offspring, we generated G607S mutant mice with motor neuron-specific expression of GFP by crossing with Hb9::eGFP mice [35].


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