TY - JOUR
T1 - Efficient recombinase-mediated cassette exchange in hPSCs to study the hepatocyte lineage reveals AAVS1 locus-mediated transgene inhibition
AU - Ordovás, Laura
AU - Boon, Ruben
AU - Pistoni, Mariaelena
AU - Chen, Yemiao
AU - Wolfs, Esther
AU - Guo, Wenting
AU - Sambathkumar, Rangarajan
AU - Bobis-Wozowicz, Sylwia
AU - Helsen, Nicky
AU - Vanhove, Jolien
AU - Berckmans, Pieter
AU - Cai, Qing
AU - Vanuytsel, Kim
AU - Eggermont, Kristel
AU - Vanslembrouck, Veerle
AU - Schmidt, Béla Z.
AU - Raitano, Susanna
AU - Van Den Bosch, Ludo
AU - Nahmias, Yaakov
AU - Cathomen, Toni
AU - Struys, Tom
AU - Verfaillie, Catherine M.
N1 - Publisher Copyright:
© 2015 The Authors.
PY - 2015/11/10
Y1 - 2015/11/10
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84946714665&partnerID=8YFLogxK
U2 - 10.1016/j.stemcr.2015.09.004
DO - 10.1016/j.stemcr.2015.09.004
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C2 - 26455413
AN - SCOPUS:84946714665
SN - 2213-6711
VL - 5
SP - 918
EP - 931
JO - Stem Cell Reports
JF - Stem Cell Reports
IS - 5
ER -