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.
An endogenously tagged fluorescent fusion protein library in mouse embryonic stem cells
Authors: Harikumar A, Edupuganti RR, Sorek M, Azad GK, Markoulaki S, Sehnalová P, Legartová S, Bártová E, Farkash-Amar S, Jaenisch R, Alon U and Meshorer E
Year of publication: 2017
Journal: Stem Cell Reports, Volume 9, Issue 4, 10 October 2017, Pages 1304-1314
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