Mutations in the Dmd gene encoding the membrane protein dystrophin are associated with the development of severe X-linked muscle diseases Duchenne and Becker myodystrophy. At the same time, along with the classic symptoms of striated muscles, dystrophin mutations can lead to a decrease in cognitive functions and behavioral abnormalities. Objective: conducting a pilot analysis of behavioral and cognitive characteristics in mice with a genetic defect that reproduces the phenotype of Duchenne myodystrophy. Materials and methods. To assess the features of motor functions and behavior, DmdDel8-34(n=13) and control animals of the wild type (n=12) were subjected to research in the tests "Load retention", "Rotarod", "Elevated plus maze" and "Object recognition". Results. It was found that mice carrying the DmdDel8-34 mutation are characterized by a decrease in motor functions, show signs of anxiety, and also show low exploratory activity. The detected features of the cognitive and emotional status are consistent with clinical observations indicating an increased risk of developing autism spectrum disorders and obsessive-compulsive disorder in patients with Dmd gene mutations. Thus, it was proved that from the 8th week of life, mice of the DmdDel8-34 line show a decrease in performance in the Rotarod and Load Retention tests. Behavioral testing in the Elevated Plus Maze test revealed a decrease in time reaching a statistically significant difference compared to the wild-type control at week 12, and when assessing cognitive functions in the Object Recognition test, it was shown that DmdDel8-34mice show an increase in the discrimination index value, which is a sign of increased efficiency of hippocampal memory. Conclusion. The study made it possible to identify some behavioral anomalies in genetically modified mice carrying a large deletion of exons 8-34 of the Dmd gene encoding dystrophin.
α-synuclein is one of the key molecular links in the pathogenesis of Parkinson’s disease. The accumulated data indicate that pathogenic mutations in the Snca gene are associated with the development of neurodegenerative brain damage, indicating the relevance of studying the synuclein neurobiological role.The aim of the study was to create a genetically modified clone of mouse stem cells with a conditional knockout of humanized α-synuclein, which can be used for the reinjection into mouse blastocysts, as well as for basic and applied in vitro research in the field of pathophysiology and neuropharmacology.Materials and methods. To create mouse stem cells with a conditional knockout of the humanized Snca gene, a previously obtained clone with the first Snca exon flanked by LoxP sites, was used. The CRISPR/Cas9-mediated homologous recombination system with donor DNA oligonucleotides of the human sites of the corresponding gene sites was used to humanize the fourth and fifth exons. Cas9 nuclease, single guide RNA, and donor DNA were transfected into mouse cells.Results. An approach to obtaining clones of mouse genetically modified stem cells expressing pathological humanized α-synuclein, has been proposed and implemented. The resulting clones were plated on Petri dishes for propagation and a further genetic analysis. Clone 126-2F4 was found out carrying the necessary genetic modifications. The results obtained are fundamentally important not only for understanding the development of the pathological process in α-synucleinopathies, but which is more important, for the development of new therapeutic approaches that will stop the extension of the human α-synuclein aggregation pathology throughout the nervous system, and the validation of these approaches in preclinical trials.Conclusion. As a result of the study, a strategy for CRISPR/Cas9-assisted homologous recombination in the genome of mouse embryonic stem cells has been developed to create a fully humanized Snca gene encoding α-synuclein, and the clone genome of mouse embryonic stem cells has been edited using a CRISPR technology. The RNA and DNA oligonucleotides necessary for the creation of RNP complexes that carry out a directed homologous recombination in the Snca locus of the mouse genome have been synthesized. The developed cell clone can serve to create a line of genetically modified mice that serve as a test system for pathophysiological and neuropharmacological studies associated with synucleinopathies. Herewith, before the induction of the Cre-dependent recombination, this line is a representative model for studying a biological role of mutant Snca. At the same time, after a Cre-dependent knockout activation, it is possible to imitate the pharmacological inhibition of α-synuclein, which is of particular interest for applied research in neuropharmacology.
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