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.
Here, we analyze the data on genetic factors involved in developing endometriosis available in current publications. To date, the genome-wide associative studies (GWAS) have revealed more than 190 loci associated with endometriosis development, however, only few polymorphisms were associated with this disease identified in two GWAS (rs1537377 CDKN2B-AS1, rs71575922 SYNE1, rs11674184 GREB1, rs1903068 KDR, rs2235529 WNT4, rs7412010 CDC42), and only one rs12700667 TSEN15P3/MIR148A polymorphic locus in four studies. Several polymorphisms located in the region of two genes GREB1 (rs11674184, rs13394619, rs35417544) and WNT4 (rs2235529, rs12037376, rs7521902) are associated with endometriosis in several GWAS. The association of 5 polymorphic loci with endometriosis was confirmed in two or more replication studies: rs13394619 GREB1, rs7521902 MIR4418/WNT4, rs1250248 FN1 and rs6542095 CKAP2L/IL1A – in two studies, rs12700667 TSEN15P3/MIR148A – in four studies. At the same time, the relationship between the vast majority of GWAS-significant polymorphic loci (more than 95 %) and endometriosis has not been confirmed in other independent studies, necessitating a need to continue endometriosis-related genetic studies, including those aimed at confirming previously identified associations.
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