Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs
Inducible loss of gene function experiments are necessary to uncover mechanisms underlying development, physiology and disease. However, current methods are complex, lack robustness and do not work in multiple cell types. Here we address these limitations by developing single-step optimized inducibl...
| Main Authors: | , , , , , , , , , , , , , , , , , , |
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| Format: | Article |
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Company of Biologists
2016
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| Online Access: | https://eprints.nottingham.ac.uk/43035/ |
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| author | Bertero, Alessandro Pawlowski, Matthias Ortmann, Daniel Snijders, Kirsten Yiangou, Loukia Cardoso de Brito, Miguel Brown, Stephanie Bernard, William G. Cooper, James D. Giacomelli, Elisa Gambardella, Laure Hannan, Nicholas R.F. Iyer, Dharini Sampaziotis, Fotios Serrano, Felipe Zonneveld, Mariëlle C.F. Sinha, Sanjay Kotter, Mark Vallier, Ludovic |
| author_facet | Bertero, Alessandro Pawlowski, Matthias Ortmann, Daniel Snijders, Kirsten Yiangou, Loukia Cardoso de Brito, Miguel Brown, Stephanie Bernard, William G. Cooper, James D. Giacomelli, Elisa Gambardella, Laure Hannan, Nicholas R.F. Iyer, Dharini Sampaziotis, Fotios Serrano, Felipe Zonneveld, Mariëlle C.F. Sinha, Sanjay Kotter, Mark Vallier, Ludovic |
| author_sort | Bertero, Alessandro |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | Inducible loss of gene function experiments are necessary to uncover mechanisms underlying development, physiology and disease. However, current methods are complex, lack robustness and do not work in multiple cell types. Here we address these limitations by developing single-step optimized inducible gene knockdown or knockout (sOPTiKD or sOPTiKO) platforms. These are based on genetic engineering of human genomic safe harbors combined with an improved tetracycline-inducible system and CRISPR/Cas9 technology. We exemplify the efficacy of these methods in human pluripotent stem cells (hPSCs), and show that generation of sOPTiKD/KO hPSCs is simple, rapid and allows tightly controlled individual or multiplexed gene knockdown or knockout in hPSCs and in a wide variety of differentiated cells. Finally, we illustrate the general applicability of this approach by investigating the function of transcription factors (OCT4 and T), cell cycle regulators (cyclin D family members) and epigenetic modifiers (DPY30). Overall, sOPTiKD and sOPTiKO provide a unique opportunity for functional analyses in multiple cell types relevant for the study of human development. |
| first_indexed | 2025-11-14T19:51:00Z |
| format | Article |
| id | nottingham-43035 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| last_indexed | 2025-11-14T19:51:00Z |
| publishDate | 2016 |
| publisher | Company of Biologists |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-430352020-05-04T18:18:23Z https://eprints.nottingham.ac.uk/43035/ Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs Bertero, Alessandro Pawlowski, Matthias Ortmann, Daniel Snijders, Kirsten Yiangou, Loukia Cardoso de Brito, Miguel Brown, Stephanie Bernard, William G. Cooper, James D. Giacomelli, Elisa Gambardella, Laure Hannan, Nicholas R.F. Iyer, Dharini Sampaziotis, Fotios Serrano, Felipe Zonneveld, Mariëlle C.F. Sinha, Sanjay Kotter, Mark Vallier, Ludovic Inducible loss of gene function experiments are necessary to uncover mechanisms underlying development, physiology and disease. However, current methods are complex, lack robustness and do not work in multiple cell types. Here we address these limitations by developing single-step optimized inducible gene knockdown or knockout (sOPTiKD or sOPTiKO) platforms. These are based on genetic engineering of human genomic safe harbors combined with an improved tetracycline-inducible system and CRISPR/Cas9 technology. We exemplify the efficacy of these methods in human pluripotent stem cells (hPSCs), and show that generation of sOPTiKD/KO hPSCs is simple, rapid and allows tightly controlled individual or multiplexed gene knockdown or knockout in hPSCs and in a wide variety of differentiated cells. Finally, we illustrate the general applicability of this approach by investigating the function of transcription factors (OCT4 and T), cell cycle regulators (cyclin D family members) and epigenetic modifiers (DPY30). Overall, sOPTiKD and sOPTiKO provide a unique opportunity for functional analyses in multiple cell types relevant for the study of human development. Company of Biologists 2016-12-01 Article PeerReviewed Bertero, Alessandro, Pawlowski, Matthias, Ortmann, Daniel, Snijders, Kirsten, Yiangou, Loukia, Cardoso de Brito, Miguel, Brown, Stephanie, Bernard, William G., Cooper, James D., Giacomelli, Elisa, Gambardella, Laure, Hannan, Nicholas R.F., Iyer, Dharini, Sampaziotis, Fotios, Serrano, Felipe, Zonneveld, Mariëlle C.F., Sinha, Sanjay, Kotter, Mark and Vallier, Ludovic (2016) Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs. Development, 143 (23). pp. 4405-4418. ISSN 1477-9129 Human pluripotent stem cells shRNA CRISPR/Cas9 OCT4 POU5F1 T brachyury DPY30 http://dev.biologists.org/content/143/23/4405 doi:10.1242/dev.138081 doi:10.1242/dev.138081 |
| spellingShingle | Human pluripotent stem cells shRNA CRISPR/Cas9 OCT4 POU5F1 T brachyury DPY30 Bertero, Alessandro Pawlowski, Matthias Ortmann, Daniel Snijders, Kirsten Yiangou, Loukia Cardoso de Brito, Miguel Brown, Stephanie Bernard, William G. Cooper, James D. Giacomelli, Elisa Gambardella, Laure Hannan, Nicholas R.F. Iyer, Dharini Sampaziotis, Fotios Serrano, Felipe Zonneveld, Mariëlle C.F. Sinha, Sanjay Kotter, Mark Vallier, Ludovic Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs |
| title | Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs |
| title_full | Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs |
| title_fullStr | Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs |
| title_full_unstemmed | Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs |
| title_short | Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs |
| title_sort | optimized inducible shrna and crispr/cas9 platforms for in vitro studies of human development using hpscs |
| topic | Human pluripotent stem cells shRNA CRISPR/Cas9 OCT4 POU5F1 T brachyury DPY30 |
| url | https://eprints.nottingham.ac.uk/43035/ https://eprints.nottingham.ac.uk/43035/ https://eprints.nottingham.ac.uk/43035/ |