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...
| Main Authors: | , , , , , , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2015-11-01
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| Series: | Stem Cell Reports |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2213671115002660 |
| _version_ | 1819170880447578112 |
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| author | Laura Ordovás Ruben Boon Mariaelena Pistoni Yemiao Chen Esther Wolfs Wenting Guo Rangarajan Sambathkumar Sylwia Bobis-Wozowicz Nicky Helsen Jolien Vanhove Pieter Berckmans Qing Cai Kim Vanuytsel Kristel Eggermont Veerle Vanslembrouck Béla Z. Schmidt Susanna Raitano Ludo Van Den Bosch Yaakov Nahmias Toni Cathomen Tom Struys Catherine M. Verfaillie |
| author_facet | Laura Ordovás Ruben Boon Mariaelena Pistoni Yemiao Chen Esther Wolfs Wenting Guo Rangarajan Sambathkumar Sylwia Bobis-Wozowicz Nicky Helsen Jolien Vanhove Pieter Berckmans Qing Cai Kim Vanuytsel Kristel Eggermont Veerle Vanslembrouck Béla Z. Schmidt Susanna Raitano Ludo Van Den Bosch Yaakov Nahmias Toni Cathomen Tom Struys Catherine M. Verfaillie |
| author_sort | Laura Ordovás |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-22T19:42:25Z |
| format | Article |
| id | doaj.art-104293e2640a470085393893e506221e |
| institution | Directory Open Access Journal |
| issn | 2213-6711 |
| language | English |
| last_indexed | 2024-12-22T19:42:25Z |
| publishDate | 2015-11-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Stem Cell Reports |
| spelling | doaj.art-104293e2640a470085393893e506221e2022-12-21T18:14:48ZengElsevierStem Cell Reports2213-67112015-11-015591893110.1016/j.stemcr.2015.09.004Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene InhibitionLaura Ordovás0Ruben Boon1Mariaelena Pistoni2Yemiao Chen3Esther Wolfs4Wenting Guo5Rangarajan Sambathkumar6Sylwia Bobis-Wozowicz7Nicky Helsen8Jolien Vanhove9Pieter Berckmans10Qing Cai11Kim Vanuytsel12Kristel Eggermont13Veerle Vanslembrouck14Béla Z. Schmidt15Susanna Raitano16Ludo Van Den Bosch17Yaakov Nahmias18Toni Cathomen19Tom Struys20Catherine M. Verfaillie21Stem Cell Institute, KU Leuven, Leuven 3000, BelgiumStem Cell Institute, KU Leuven, Leuven 3000, BelgiumStem Cell Institute, KU Leuven, Leuven 3000, BelgiumStem Cell Institute, KU Leuven, Leuven 3000, BelgiumGroup of Morphology, Biomedical Research Institute, Hasselt University, Diepenbeek 3590, BelgiumLeuven Research Institute for Neuroscience and Disease (LIND), Leuven 3000, BelgiumStem Cell Institute, KU Leuven, Leuven 3000, BelgiumInstitute for Cell and Gene Therapy, University Medical Center Freiburg, Freiburg 79108, GermanyStem Cell Institute, KU Leuven, Leuven 3000, BelgiumStem Cell Institute, KU Leuven, Leuven 3000, BelgiumStem Cell Institute, KU Leuven, Leuven 3000, BelgiumStem Cell Institute, KU Leuven, Leuven 3000, BelgiumStem Cell Institute, KU Leuven, Leuven 3000, BelgiumStem Cell Institute, KU Leuven, Leuven 3000, BelgiumStem Cell Institute, KU Leuven, Leuven 3000, BelgiumSwitch Laboratory, VIB, Leuven 3000, BelgiumStem Cell Institute, KU Leuven, Leuven 3000, BelgiumLeuven Research Institute for Neuroscience and Disease (LIND), Leuven 3000, BelgiumDepartment of Cell and Developmental Biology, Hebrew University of Jerusalem, Jerusalem 91904, IsraelInstitute for Cell and Gene Therapy, University Medical Center Freiburg, Freiburg 79108, GermanyGroup of Morphology, Biomedical Research Institute, Hasselt University, Diepenbeek 3590, BelgiumStem Cell Institute, KU Leuven, Leuven 3000, BelgiumTools 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.http://www.sciencedirect.com/science/article/pii/S2213671115002660 |
| spellingShingle | Laura Ordovás Ruben Boon Mariaelena Pistoni Yemiao Chen Esther Wolfs Wenting Guo Rangarajan Sambathkumar Sylwia Bobis-Wozowicz Nicky Helsen Jolien Vanhove Pieter Berckmans Qing Cai Kim Vanuytsel Kristel Eggermont Veerle Vanslembrouck Béla Z. Schmidt Susanna Raitano Ludo Van Den Bosch Yaakov Nahmias Toni Cathomen Tom Struys Catherine M. Verfaillie Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition Stem Cell Reports |
| 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 |
| url | http://www.sciencedirect.com/science/article/pii/S2213671115002660 |
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