A modular open platform for systematic functional studies under physiological conditions

Any profound comprehension of gene function requires detailed information about the subcellular localization, molecular interactions and spatiotemporal 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 specificmutations 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.