Overcoming the Silencing of Doxycycline-Inducible Promoters in hiPSC-derived Cardiomyocytes [version 1; peer review: 1 approved, 2 approved with reservations]
Background Human induced pluripotent stem cells (hiPSCs) are pivotal for studying human development, modeling diseases, and advancing regenerative medicine. Effective control of transgene expression is crucial to achieve temporal and quantitative precision in all of these contexts. The doxycycline (...
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F1000 Research Ltd
2024-12-01
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| author | Michelle Guichardaz Elisa Balmas Alessandro Bertero Sveva Bottini |
| author_facet | Michelle Guichardaz Elisa Balmas Alessandro Bertero Sveva Bottini |
| author_sort | Michelle Guichardaz |
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| description | Background Human induced pluripotent stem cells (hiPSCs) are pivotal for studying human development, modeling diseases, and advancing regenerative medicine. Effective control of transgene expression is crucial to achieve temporal and quantitative precision in all of these contexts. The doxycycline (dox)-inducible OPTi-OX system, which integrates the Tet-On 3G transactivator and dox-responsive transgene at the hROSA26 and AAVS1 genomic safe harbors (GSHs), respectively, offers a promising solution. Yet, transgene silencing, particularly in hiPSC-derived cardiomyocytes (hiPSC-CMs), limits its utility. Methods To address this, we evaluated strategies to enhance dox-inducible transgene expression. We compared two promoters, TRE3VG and T11, for activity and stability, and investigated the addition of a Ubiquitous Chromatin Opening Element (UCOE) to reduce silencing. We also tested relocating the transgene cassette to the CLYBL GSH, and employed sodium butyrate (SB), a histone deacetylase inhibitor, to restore promoter activity. Transgene expression was assessed via flow cytometry and real-time quantitative PCR. Results TRE3VG exhibited higher activity than T11, but both were prone to silencing. UCOE did not enhance promoter activity in hiPSCs, but modestly reduced silencing in hiPSC-CMs. Targeting the CLYBL locus improved promoter activity compared to AAVS1 in both hiPSCs and hiPSC-CMs. SB restored activity in silenced inducible promoters within hiPSC-CMs, but compromised hiPSC viability. Unexpectedly, Tet-On 3G was silenced in some clones and could not be reactivated by SB. Conclusions These findings underscore the need for integrating multiple strategies, including careful GSH selection, improved cassette design, epigenetic modulation, and clone screening, to develop robust dox-inducible systems that retain functionality during hiPSC differentiation. |
| format | Article |
| id | doaj-art-386f6494b35f44e3904863ca8e00d197 |
| institution | Kabale University |
| issn | 2732-5121 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | F1000 Research Ltd |
| record_format | Article |
| series | Open Research Europe |
| spelling | doaj-art-386f6494b35f44e3904863ca8e00d1972025-02-07T01:00:00ZengF1000 Research LtdOpen Research Europe2732-51212024-12-01420586Overcoming the Silencing of Doxycycline-Inducible Promoters in hiPSC-derived Cardiomyocytes [version 1; peer review: 1 approved, 2 approved with reservations]Michelle Guichardaz0Elisa Balmas1https://orcid.org/0000-0002-4600-6809Alessandro Bertero2https://orcid.org/0000-0002-4919-9087Sveva Bottini3Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center “Guido Tarone”, University of Turin, Torino, 10126, ItalyDepartment of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center “Guido Tarone”, University of Turin, Torino, 10126, ItalyDepartment of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center “Guido Tarone”, University of Turin, Torino, 10126, ItalyDepartment of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center “Guido Tarone”, University of Turin, Torino, 10126, ItalyBackground Human induced pluripotent stem cells (hiPSCs) are pivotal for studying human development, modeling diseases, and advancing regenerative medicine. Effective control of transgene expression is crucial to achieve temporal and quantitative precision in all of these contexts. The doxycycline (dox)-inducible OPTi-OX system, which integrates the Tet-On 3G transactivator and dox-responsive transgene at the hROSA26 and AAVS1 genomic safe harbors (GSHs), respectively, offers a promising solution. Yet, transgene silencing, particularly in hiPSC-derived cardiomyocytes (hiPSC-CMs), limits its utility. Methods To address this, we evaluated strategies to enhance dox-inducible transgene expression. We compared two promoters, TRE3VG and T11, for activity and stability, and investigated the addition of a Ubiquitous Chromatin Opening Element (UCOE) to reduce silencing. We also tested relocating the transgene cassette to the CLYBL GSH, and employed sodium butyrate (SB), a histone deacetylase inhibitor, to restore promoter activity. Transgene expression was assessed via flow cytometry and real-time quantitative PCR. Results TRE3VG exhibited higher activity than T11, but both were prone to silencing. UCOE did not enhance promoter activity in hiPSCs, but modestly reduced silencing in hiPSC-CMs. Targeting the CLYBL locus improved promoter activity compared to AAVS1 in both hiPSCs and hiPSC-CMs. SB restored activity in silenced inducible promoters within hiPSC-CMs, but compromised hiPSC viability. Unexpectedly, Tet-On 3G was silenced in some clones and could not be reactivated by SB. Conclusions These findings underscore the need for integrating multiple strategies, including careful GSH selection, improved cassette design, epigenetic modulation, and clone screening, to develop robust dox-inducible systems that retain functionality during hiPSC differentiation.https://open-research-europe.ec.europa.eu/articles/4-266/v1Human induced pluripotent stem cells cardiomyocytes inducible expression silencing TRE3VG promoter T11 promotereng |
| spellingShingle | Michelle Guichardaz Elisa Balmas Alessandro Bertero Sveva Bottini Overcoming the Silencing of Doxycycline-Inducible Promoters in hiPSC-derived Cardiomyocytes [version 1; peer review: 1 approved, 2 approved with reservations] Open Research Europe Human induced pluripotent stem cells cardiomyocytes inducible expression silencing TRE3VG promoter T11 promoter eng |
| title | Overcoming the Silencing of Doxycycline-Inducible Promoters in hiPSC-derived Cardiomyocytes [version 1; peer review: 1 approved, 2 approved with reservations] |
| title_full | Overcoming the Silencing of Doxycycline-Inducible Promoters in hiPSC-derived Cardiomyocytes [version 1; peer review: 1 approved, 2 approved with reservations] |
| title_fullStr | Overcoming the Silencing of Doxycycline-Inducible Promoters in hiPSC-derived Cardiomyocytes [version 1; peer review: 1 approved, 2 approved with reservations] |
| title_full_unstemmed | Overcoming the Silencing of Doxycycline-Inducible Promoters in hiPSC-derived Cardiomyocytes [version 1; peer review: 1 approved, 2 approved with reservations] |
| title_short | Overcoming the Silencing of Doxycycline-Inducible Promoters in hiPSC-derived Cardiomyocytes [version 1; peer review: 1 approved, 2 approved with reservations] |
| title_sort | overcoming the silencing of doxycycline inducible promoters in hipsc derived cardiomyocytes version 1 peer review 1 approved 2 approved with reservations |
| topic | Human induced pluripotent stem cells cardiomyocytes inducible expression silencing TRE3VG promoter T11 promoter eng |
| url | https://open-research-europe.ec.europa.eu/articles/4-266/v1 |
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