An Endogenously Tagged Fluorescent Fusion Protein Library in Mouse Embryonic Stem Cells
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Author(s)
Harikumar, Arigela
Edupuganti, Raghu Ram
Sorek, Matan
Azad, Gajendra Kumar
Markoulaki, Styliani
Sehnalová, Petra
Legartová, Soňa
Bártová, Eva
Farkash-Amar, Shlomit
Jaenisch, Rudolf
Date Issued
September 2017
Journal
Stem Cell Reports
Publisher
Elsevier
Citation
Harikumar, Arigela et al. “An Endogenously Tagged Fluorescent Fusion Protein Library in Mouse Embryonic Stem Cells.” Stem Cell Reports 9, 4 (October 2017): 1304–1314 © 2017 The Authors
Version
Final published version
Abstract
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. Keywords: embryonic stem cells; imaging; live imaging; fluorescence; differentiation; pluripotency; GFP; microscopy; DNA damage; protein dynamics
MIT Department
Massachusetts Institute of Technology. Department of Biology
Terms of Use
Creative Commons Attribution-NonCommercial-NoDerivs License
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DOI of Published Version
https://doi.org/10.1016/J.STEMCR.2017.08.022
