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

S. V. Pavlova; Павлова С. В.; K. R. Valetdinova; Валетдинова К. Р.; T. B. Malankhanova; Маланханова Т. Б.; D. E. Polivtsev; Поливцев Д. Е.; A. A. Malahova; Малахова А. А.; E. V. Grigor’eva; Григорьева Е. В.; A. I. Shevchenko; Шевченко А. И.; S. M. Zakian; Закиян С. М.; S. P. Medvedev; Медведев С. П.

doi:10.31857/S047514502306006X

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

Authors: Pavlova S.V.¹^,2^,3, Valetdinova K.R.¹, Malankhanova T.B.¹, Polivtsev D.E.¹^,4, Malahova A.A.¹^,2^,3, Grigor’eva E.V.¹^,2^,3, Shevchenko A.I.¹^,2^,3, Zakian S.M.¹^,2^,3, Medvedev S.P.¹^,2^,3
Affiliations:
1. Federal Research Center Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences
2. Meshalkin National Medical Research Center of the Ministry of Health of the Russian Federation
3. Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences
4. Novosibirsk State University
Issue: Vol 54, No 6 (2023)
Pages: 415-428
Section: Collection of pluripotent stem cell lines
URL: https://journal-vniispk.ru/0475-1450/article/view/232038
DOI: https://doi.org/10.31857/S047514502306006X
EDN: https://elibrary.ru/GESSIW
ID: 232038

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Abstract
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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 marker 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.

Keywords

induced pluripotent stem cells (iPSCs), transgenesis, CRISPR/Cas9 system, AsCas12a (AsCpf1) nuclease, isogenic cell lines

About the authors

S. V. Pavlova

Federal Research Center Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences; Meshalkin National Medical Research Center of the Ministry of Health of the Russian Federation; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences

Email: medvedev@bionet.nsc.ru
Russia, 630090, Novosibirsk; Russia, 630055, Novosibirsk; Russia, 630090, Novosibirsk

References

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