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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...

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Autores principales: 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.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2015
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.
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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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