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Mulholland, Christopher B.; Smets, Martha; Schmidtmann, Elisabeth; Leidescher, Susanne; Markaki, Yolanda; Hofweber, Mario; Qin, Weihua; Manzo, Massimiliano; Kremmer, Elisabeth; Thanisch, Katharina; Bauer, Christina; Rombaut, Pascaline; Herzog, Franz; Leonhardt, Heinrich and Bultmann, Sebastian (2015): A modular open platform for systematic functional studies under physiological conditions. In: Nucleic Acids Research, Vol. 43, No. 17, e112 [PDF, 15MB]

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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.

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