Technique for Animal Knockout system by Electroporation (TAKE) is a simple and efficient method to generate genetically modified (GM) mice using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems. To reinforce the versatility of electroporation used for gene editing in mice, the electric condition was optimized for vitrified-warmed mouse embryos, and applied to the fresh embryos from widely used inbred strains (C57BL/6NCr, BALB/cCrSlc, FVB/NJcl, and C3H/HeJJcl). The electric pulse settings (poring pulse: voltage, 150 V; pulse width, 1.0 ms; pulse interval, 50 ms; number of pulses, +4; transfer pulse: voltage, 20 V; pulse width, 50 ms; pulse interval, 50 ms; number of pulses, ±5) were optimal for vitrified-warmed mouse embryos, which could efficiently deliver the gRNA/Cas9 complex into the zygotes without zona pellucida thinning process and edit the target locus. These electric condition efficiently generated GM mice in widely used inbred mouse strains. In addition, electroporation using the electrode with a 5 mm gap could introduce more than 100 embryos within 5 min without specific pretreatment and sophisticated technical skills, such as microinjection, and exhibited a high developmental rate of embryos and genomeediting efficiency in the generated offspring, leading to the rapid and efficient generation of genome editing mice. The electric condition used in this study is highly versatile and can contribute to understanding human diseases and gene functions by generating GM mice more easily and efficiently.

Duchenne muscular dystrophy (DMD), the most severe form of dystrophinopathies, is a fatal X-linked recessive neuromuscular disorder characterized by progressive muscle degeneration and various extents of intellectual disabilities. Physiological and pathological roles of the responsible gene, dystrophin, in the brain remain elusive due to the presence of multiple dystrophin products, mainly full-length dystrophin, Dp427, and the short product, Dp71. In this study, we generated a Dp71-speci c hemagglutinin (HA) peptide tag-insertion mice to enable speci c detection of intrinsic Dp71 expression by anti-HA tag antibodies. Immunohistochemical detections in the transgenic mice demonstrated Dp71 expression not only at the blood-brain barrier, where astrocytic endfeet surround the microvessels, but also at the inhibitory postsynapse of hippocampal dentate granule neurons. Interestingly, hippocampal cornu ammonis (CA)1 pyramidal neurons were negative for Dp71 although Dp427 detected by anti-dystrophin antibody was clearly present at the inhibitory postsynapse, suggesting cell-type dependent dystrophin expressions. Precise examination using the primary hippocampal culture validated exclusive localization of Dp71 at the inhibitory postsynaptic compartment but not at the excitatory synapse in neurons. We further performed interactome analysis and found that Dp71 formed distinct molecular complexes, i.e. synapse-associated Dp71 interacted with dystroglycan (Dg) and dystrobrevinb (Dtnb) whereas glia-associated Dp71 did with Dg and dystrobrevina (Dtna). Thus, our data indicates that Dp71 and its binding partners are relevant to the inhibitory postsynaptic function of hippocampal granule neurons and the novel Dp71-transgenic mouse provides a valuable tool to understand precise physiological expressions and functions of Dp71 and its interaction proteins in vivo and in vitro.

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Duchenne muscular dystrophy (DMD), the most severe form of dystrophinopathies, is a fatal X-linked recessive neuromuscular disorder characterized by progressive muscle degeneration and various extents of intellectual disabilities. Physiological and pathological roles of the responsible gene, dystrophin, in the brain remain elusive due to the presence of multiple dystrophin products, mainly full-length dystrophin, Dp427, and the short product, Dp71. In this study, we generated a Dp71-specific hemagglutinin (HA) peptide tag-insertion mice to enable specific detection of intrinsic Dp71 expression by anti-HA tag antibodies. Immunohistochemical detections in the transgenic mice demonstrated Dp71 expression not only at the blood-brain barrier, where astrocytic endfeet surround the microvessels, but also at the inhibitory postsynapse of hippocampal dentate granule neurons. Interestingly, hippocampal cornu ammonis (CA)1 pyramidal neurons were negative for Dp71 although Dp427 detected by anti-dystrophin antibody was clearly present at the inhibitory postsynapse, suggesting cell-type dependent dystrophin expressions. Precise examination using the primary hippocampal culture validated exclusive localization of Dp71 at the inhibitory postsynaptic compartment but not at the excitatory synapse in neurons. We further performed interactome analysis and found that Dp71 formed distinct molecular complexes, i.e. synapse-associated Dp71 interacted with dystroglycan (Dg) and dystrobrevinb (Dtnb) whereas glia-associated Dp71 did with Dg and dystrobrevina (Dtna). Thus, our data indicates that Dp71 and its binding partners are relevant to the inhibitory postsynaptic function of hippocampal granule neurons and the novel Dp71-transgenic mouse provides a valuable tool to understand precise physiological expressions and functions of Dp71 and its interaction proteins in vivo and in vitro.