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Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition
Tools for rapid and efficient transgenesis in “safe harbor” loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that all...
Autores principales: | , , , , , , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4649136/ https://www.ncbi.nlm.nih.gov/pubmed/26455413 http://dx.doi.org/10.1016/j.stemcr.2015.09.004 |
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author | Ordovás, Laura Boon, Ruben Pistoni, Mariaelena Chen, Yemiao Wolfs, Esther Guo, Wenting Sambathkumar, Rangarajan Bobis-Wozowicz, Sylwia Helsen, Nicky Vanhove, Jolien Berckmans, Pieter Cai, Qing Vanuytsel, Kim Eggermont, Kristel Vanslembrouck, Veerle Schmidt, Béla Z. Raitano, Susanna Van Den Bosch, Ludo Nahmias, Yaakov Cathomen, Toni Struys, Tom Verfaillie, Catherine M. |
author_facet | Ordovás, Laura Boon, Ruben Pistoni, Mariaelena Chen, Yemiao Wolfs, Esther Guo, Wenting Sambathkumar, Rangarajan Bobis-Wozowicz, Sylwia Helsen, Nicky Vanhove, Jolien Berckmans, Pieter Cai, Qing Vanuytsel, Kim Eggermont, Kristel Vanslembrouck, Veerle Schmidt, Béla Z. Raitano, Susanna Van Den Bosch, Ludo Nahmias, Yaakov Cathomen, Toni Struys, Tom Verfaillie, Catherine M. |
author_sort | Ordovás, Laura |
collection | PubMed |
description | Tools for rapid and efficient transgenesis in “safe harbor” loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes. |
format | Online Article Text |
id | pubmed-4649136 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Elsevier |
record_format | MEDLINE/PubMed |
spelling | pubmed-46491362015-12-11 Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition Ordovás, Laura Boon, Ruben Pistoni, Mariaelena Chen, Yemiao Wolfs, Esther Guo, Wenting Sambathkumar, Rangarajan Bobis-Wozowicz, Sylwia Helsen, Nicky Vanhove, Jolien Berckmans, Pieter Cai, Qing Vanuytsel, Kim Eggermont, Kristel Vanslembrouck, Veerle Schmidt, Béla Z. Raitano, Susanna Van Den Bosch, Ludo Nahmias, Yaakov Cathomen, Toni Struys, Tom Verfaillie, Catherine M. Stem Cell Reports Resource Tools for rapid and efficient transgenesis in “safe harbor” loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes. Elsevier 2015-10-08 /pmc/articles/PMC4649136/ /pubmed/26455413 http://dx.doi.org/10.1016/j.stemcr.2015.09.004 Text en © 2015 The Authors http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Resource Ordovás, Laura Boon, Ruben Pistoni, Mariaelena Chen, Yemiao Wolfs, Esther Guo, Wenting Sambathkumar, Rangarajan Bobis-Wozowicz, Sylwia Helsen, Nicky Vanhove, Jolien Berckmans, Pieter Cai, Qing Vanuytsel, Kim Eggermont, Kristel Vanslembrouck, Veerle Schmidt, Béla Z. Raitano, Susanna Van Den Bosch, Ludo Nahmias, Yaakov Cathomen, Toni Struys, Tom Verfaillie, Catherine M. Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition |
title | Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition |
title_full | Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition |
title_fullStr | Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition |
title_full_unstemmed | Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition |
title_short | Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition |
title_sort | efficient recombinase-mediated cassette exchange in hpscs to study the hepatocyte lineage reveals aavs1 locus-mediated transgene inhibition |
topic | Resource |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4649136/ https://www.ncbi.nlm.nih.gov/pubmed/26455413 http://dx.doi.org/10.1016/j.stemcr.2015.09.004 |
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