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Transgenic Lines of Human Induced Pluripotent Stem Cells ICGi022-A-6 and ICGi022-A-7 with Doxycycline-Inducible Variants of Programmable Nuclease AsCas12a

  • COLLECTION OF PLURIPOTENT STEM CELL LINES
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Abstract

Genome editing in human pluripotent stem cells using programmable nucleases makes it possible to create models of hereditary pathologies using directed transgenesis, gene knockout, and replacement of individual nucleotides in DNA sequences. Using CRISPR/SpCas9-mediated homologous recombination at the AAVS1 locus, clones of human induced pluripotent stem cells (iPSCs) ICGi022-A (Malakhova et al., 2020) were obtained, which carry transgenes of two variants of the nuclease AsCas12a (also known as AsCpf1), recognizing different PAM consensuses, and the reverse doxycycline transgene-dependent transactivator M2rtTA. For each AsCas12a variant, the lines ICGi022-A-6 (AsCas12a, PAM 5'-TTTV-3') and ICGi022-A-7 (AsCas12a, PAM 5'-TYCV-3') were obtained. Using Western blot analysis, it was shown that the addition of doxycycline to the culture medium causes activation of the expression of AsCas12a(TTTV) and AsCas12a(TYCV) proteins. The resulting transgenic iPSC clones were subjected to molecular and cytogenetic analysis. Using quantitative PCR and immunocytochemical analysis, it was shown that they have a high level of mRNA expression of gene markers of pluripotent cells, namely OCT4, NANOG, and SOX2, as well as specific expression of protein markers OCT4, SOX2, SSEA-4, and TRA-1-60. In addition, using iPSCs spontaneous differentiation into embryoid bodies, it was found that transgenic clones can give derivatives of all three primitive germ layers: ectoderm, mesoderm, and endoderm. Cytogenetic analysis showed that transgenic iPSC clones have a normal karyotype, 46,XX.

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ACKNOWLEDGMENTS

Analysis of immunofluorescent staining was carried out using the resources of the Center for Microscopic Analysis of Biological Objects of the Institute of Cytology and Genetics (Siberian Branch, Russian Academy of Sciences), supported by the Budget Project of the Institute of Cytology and Genetics FWNR-2022-0015.

Funding

The study was supported by the Ministry of Science and Higher Education of the Russian Federation, agreement no. 075-15-2021-1063 dated September 28, 2021.

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Authors and Affiliations

  1. Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences, 630090, Novosibirsk, Russia

    S. V. Pavlova, K. R. Valetdinova, T. B. Malankhanova, D. E. Polivtsev, A. A. Malahova, E. V. Grigor’eva, A. I. Shevchenko, S. M. Zakian & S. P. Medvedev

  2. Meshalkin National Medical Research Center, Ministry of Health of the Russian Federation, 630055, Novosibirsk, Russia

    S. V. Pavlova, A. A. Malahova, E. V. Grigor’eva, A. I. Shevchenko, S. M. Zakian & S. P. Medvedev

  3. Novosibirsk State University, 630090, Novosibirsk, Russia

    D. E. Polivtsev

  4. Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, 630090, Novosibirsk, Russia

    S. V. Pavlova, A. A. Malahova, E. V. Grigor’eva, A. I. Shevchenko, S. M. Zakian & S. P. Medvedev

Authors
  1. S. V. Pavlova
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  2. K. R. Valetdinova
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  3. T. B. Malankhanova
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  4. D. E. Polivtsev
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  5. A. A. Malahova
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  6. E. V. Grigor’eva
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  7. A. I. Shevchenko
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  8. S. M. Zakian
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  9. S. P. Medvedev
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Contributions

S.V. Pavlova, S.M. Zakiyan, and S.P. Medvedev: experimental design, data analysis, and article writing. S.P. Medvedev and A.I. Shevchenko: creation of donor constructs carrying AsCas12a transgenes. K.R. Valetdinova and T.B. Malankhanova: transgenesis in iPSCs using the CRISPR/Cas9 system and donor constructs, selection of iPSC clones resistant to neomycin and puromycin, DNA analysis of clones for the presence of transgene insertion using PCR and Western blot. D.E. Polivtsev and A.A. Malakhova, E.V. Grigorieva: characteristics of iPSCs obtained as a result of lines editing.

Corresponding author

Correspondence to S. P. Medvedev.

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CONFLICT OF INTEREST

The authors of this work declare that they have no conflicts of interest.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

The study was approved by the ethical commission of the Federal Center for Neurosurgery of the Ministry of Health of the Russian Federation, Novosibirsk, protocol no. 1 of March 14, 2017. Patients gave voluntary informed consent.

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Passport of Pluripotent Stem Cell Lines

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Pavlova, S.V., Valetdinova, K.R., Malankhanova, T.B. et al. Transgenic Lines of Human Induced Pluripotent Stem Cells ICGi022-A-6 and ICGi022-A-7 with Doxycycline-Inducible Variants of Programmable Nuclease AsCas12a. Russ J Dev Biol 54, 374–386 (2023). https://doi.org/10.1134/S1062360423060061

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  • DOI: https://doi.org/10.1134/S1062360423060061

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