Embryonic stem cells (ESCs), with their dual capacity to self-renew and differentiate, are commonly used to study differentiation, epigenetic regulation, lineage choices, and more. Using non-directed retroviral integration of a YFP/Cherry exon into mouse ESCs, we generated a library of over 200 endogenously tagged fluorescent fusion proteins and present several proof-of-concept applications of this library. We show the utility of this library to track proteins in living cells; screen for pluripotency-related factors; identify heterogeneously expressing proteins; measure the dynamics of endogenously labeled proteins; track proteins recruited to sites of DNA damage; pull down tagged fluorescent fusion proteins using anti-Cherry antibodies; and test for interaction partners. Thus, this library can be used in a variety of different directions, either exploiting the fluorescent tag for imaging-based techniques or utilizing the fluorescent fusion protein for biochemical pull-down assays, including immunoprecipitation, co-immunoprecipitation, chromatin immunoprecipitation, and more.

Department of Genetics The Alexander Silberman Institute of Life Sciences The Hebrew University of Jerusalem Jerusalem 91904 Israel

Department of Genetics The Alexander Silberman Institute of Life Sciences The Hebrew University of Jerusalem Jerusalem 91904 Israel; The Edmond and Lily Safra Center for Brain Sciences The Hebrew University of Jerusalem Jerusalem 91904 Israel

Department of Molecular Cell Biology Weizmann Institute of Science Rehovot 76100 Israel

Institute of Biophysics Academy of Sciences of the Czech Republic v v i Královopolská 135 612 65 Brno Czech Republic

Whitehead Institute for Biomedical Research Cambridge MA USA

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