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Nucleic Acids Res. 2015 Sep 30;43(17):e112. doi: 10.1093/nar/gkv550. Epub 2015 May 24.

A modular open platform for systematic functional studies under physiological conditions.

Author information

1
Ludwig Maximilians University Munich, Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Großhaderner Strasse 2, 82152 Planegg-Martinsried, Germany.
2
Helmholtz Center Munich, German Research Center for Environmental Health (GmbH), Institute of Molecular Immunology, Marchioninistrasse 25, 81377 Munich, Germany.
3
Gene Center and Department of Biochemistry, Ludwig Maximilians University Munich, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.
4
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.
5
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.

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.

PMID:
26007658
PMCID:
PMC4787826
DOI:
10.1093/nar/gkv550
[Indexed for MEDLINE]
Free PMC Article

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