Publication Date:
2015-09-30
Description:
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.
Keywords:
Synthetic Biology and Assembly Cloning, Targeted gene modification
Print ISSN:
0305-1048
Electronic ISSN:
1362-4962
Topics:
Biology
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