title: A modular open platform for systematic functional studies under physiological conditions.
abstract: Any profound comprehension of gene function requires detailed information about the subcellular localization, molecular interactions and spatio-temporal dynamics of gene products. We developed a multifunctional integrase (MIN) tag for rapid and versatile genome engineering that serves not only as a genetic entry site for the Bxb1 integrase but also as a novel epitope tag for standardized detection and precipitation. For the systematic study of epigenetic factors, including Dnmt1, Dnmt3a, Dnmt3b, Tet1, Tet2, Tet3 and Uhrf1, we generated MIN-tagged embryonic stem cell lines and created a toolbox of prefabricated modules that can be integrated via Bxb1-mediated recombination. We used these functional modules to study protein interactions and their spatio-temporal dynamics as well as gene expression and specific mutations during cellular differentiation and in response to external stimuli. Our genome engineering strategy provides a versatile open platform for efficient generation of multiple isogenic cell lines to study gene function under physiological conditions.
authors.affiliation: Ludwig Maximilians University Munich, Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Großhaderner Strasse 2, 82152 Planegg-Martinsried, Germany.
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Mulholland
authors.affiliation: Ludwig Maximilians University Munich, Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Großhaderner Strasse 2, 82152 Planegg-Martinsried, Germany.
authors.forename: Martha
authors.initials: M
authors.lastname: Smets
authors.affiliation: Ludwig Maximilians University Munich, Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Großhaderner Strasse 2, 82152 Planegg-Martinsried, Germany.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Schmidtmann
authors.affiliation: Ludwig Maximilians University Munich, Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Großhaderner Strasse 2, 82152 Planegg-Martinsried, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Leidescher
authors.affiliation: Ludwig Maximilians University Munich, Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Großhaderner Strasse 2, 82152 Planegg-Martinsried, Germany.
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Markaki
authors.affiliation: Ludwig Maximilians University Munich, Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Großhaderner Strasse 2, 82152 Planegg-Martinsried, Germany.
authors.forename: Mario
authors.initials: M
authors.lastname: Hofweber
authors.affiliation: Ludwig Maximilians University Munich, Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Großhaderner Strasse 2, 82152 Planegg-Martinsried, Germany.
authors.forename: Weihua
authors.initials: W
authors.lastname: Qin
authors.affiliation: Ludwig Maximilians University Munich, Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Großhaderner Strasse 2, 82152 Planegg-Martinsried, Germany.
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Manzo
authors.affiliation: Helmholtz Center Munich, German Research Center for Environmental Health (GmbH), Institute of Molecular Immunology, Marchioninistrasse 25, 81377 Munich, Germany.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Kremmer
authors.affiliation: Ludwig Maximilians University Munich, Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Großhaderner Strasse 2, 82152 Planegg-Martinsried, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Thanisch
authors.affiliation: Ludwig Maximilians University Munich, Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Großhaderner Strasse 2, 82152 Planegg-Martinsried, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Bauer
authors.affiliation: Gene Center and Department of Biochemistry, Ludwig Maximilians University Munich, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.
authors.forename: Pascaline
authors.initials: P
authors.lastname: Rombaut
authors.affiliation: Gene Center and Department of Biochemistry, Ludwig Maximilians University Munich, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.
authors.forename: Franz
authors.initials: F
authors.lastname: Herzog
authors.affiliation: Ludwig Maximilians University Munich, Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Großhaderner Strasse 2, 82152 Planegg-Martinsried, Germany h.leonhardt@lmu.de.
authors.forename: Heinrich
authors.initials: H
authors.lastname: Leonhardt
authors.affiliation: Ludwig Maximilians University Munich, Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Großhaderner Strasse 2, 82152 Planegg-Martinsried, Germany bultmann@bio.lmu.de.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Bultmann
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D064113:CRISPR-Cas Systems; D002454:Cell Differentiation / Q000235:genetics; D060846:Cell Engineering / Q000379:methods*; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000378:metabolism; D015870:Gene Expression; D056426:Genetic Loci; D023281:Genomics / Q000379:methods; D019426:Integrases / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D009154:Mutation; D051381:Rats; D011995:Recombination, Genetic
chemical_list: D000911:Antibodies, Monoclonal; D019426:Integrases