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. Using a gene-tagging approach, Meshorer and colleagues describe in this article the generation of an endogenously tagged fluorescent fusion library in mouse ESCs, providing the community with over 200 YFP/Cherry-tagged clones highly expressed in ESCs. The paper describes the generation of the library as well as several potential uses and applications.
Bibliographical noteFunding Information:
This work was supported by the Israel Science Foundation ( ISF 1140/2017 to E.M. and ISF 1349/15 to U.A.); the European Research Council (to E.M.); the European Union's Horizon 2020 research and innovation program FET-OPEN ( CellViewer , no. 686637 ); and NIH grants HD045022 , R37-CA084198 , and R01 NS088538-01 (to R.J.). E.M. is the incumbent of the Arthur Gutterman Professor Chair. U.A. is the incumbent of the Abisch-Frenkel Professor Chair. R.R.E. and A.H. were supported by the Marie Curie Nucleosome4D network ; R.R.E. was supported by a post-doctoral fellowship from the Lady Davis Foundation . The authors thank Dan Lehman for help with single-cell sorting, as well as Bill Breuer, Tamar Danon, Mayan Sheiba, David Gokhman, Naveh Evantal, and Lilya Verchovsky for technical assistance. We dedicate this paper to the memory of Lilya Verchovsky.
© 2017 The Authors
- DNA damage
- embryonic stem cells
- live imaging
- protein dynamics