Abstract
The deletions and mutations in the UBE2A gene cause X-linked mental retardation syndrome of the Nascimento type first described in 2006 (Nascimento et al., 2006). At the moment, approximately two dozen missense and nonsense mutations in the UBE2A gene associated with Nascimento-type mental retardation syndrome are known (Cordeddu et al., 2020). To study the role of the UBE2A gene in neurodevelopment, the authors generated a human iPSC line with knockout of the UBE2A gene (RCPCMi009-A-1) using genome editing CRISPR/Cas9 technology. The knockout of the UBE2A gene was confirmed by Western blotting. The pluripotent state of the RCPCMi009-A-1 iPSC line was confirmed by typical stem cell morphology, normal male karyotype (46,XY) maintenance, expression of pluripotency markers, and the ability to differentiate into the derivatives of three germ layers.
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ACKNOWLEDGMENTS
We are grateful to the Center for High-Precision Genome Editing and Genetic Technologies in Biomedicine of the Lopukhin Federal Research and Clinical Center of Physicochemical Medicine for their expertise and guidance in the field of genome editing.
Funding
The research was carried out with the financial support of the Russian Science Foundation, grant no. 21-65-00017, https://rscf.ru/project/21-65-00017/.
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E.A. Khomyakova and A.V. Fedorenko carried out the cultural and molecular-genetic part of the work, namely, cloning a plasmid vector with a guide RNA, experiments on editing the iPSC genome, selection of individual iPSC clones and their detailed characterization, and spontaneous differentiation of iPSCs into derivatives of three germ layers. A.V. Surdina and L.D. Belikova analyzed the expression of pluripotency markers by immunocytochemical staining and flow cytometry, respectively. E.A. Volovikov conducted Western blotting. E.A. Zerkalenkova performed an analysis of the karyotype of the iPSC line with the knockout of the UBE2A gene. M.A. Lagarkova and A.N. Bogomazova developed the design of the experiment, analyzed the obtained data, and participated in the writing of the article.
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The research project was examined and approved on June 1, 2021, by the Ethical Commission of the Federal Scientific and Clinical Center of Physicochemical Medicine, protocol no. 1 from January 6, 2021.
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Passport of the Pluripotent Stem Cell Lines
Translated by A. Ermakov
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Khomyakova, E.A., Fedorenko, A.V., Surdina, A.V. et al. Derivation of Induced Pluripotent Stem Cells Line (RCPCMi009-A-1) with Knockout of the UBE2A Gene Using CRISPR/Cas9 Genome Editing. Russ J Dev Biol 54, 365–373 (2023). https://doi.org/10.1134/S1062360423060048
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DOI: https://doi.org/10.1134/S1062360423060048