An Endogenously Tagged Fluorescent Fusion Protein Library in Mouse Embryonic Stem Cells

Arigela Harikumar, Raghu Ram Edupuganti, Matan Sorek, Gajendra Kumar Azad, Styliani Markoulaki, Petra Sehnalová, Soňa Legartová, Eva Bártová, Shlomit Farkash-Amar, Rudolf Jaenisch, Uri Alon, Eran Meshorer*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

16 Scopus citations

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. 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.

Original languageAmerican English
Pages (from-to)1304-1314
Number of pages11
JournalStem Cell Reports
Volume9
Issue number4
DOIs
StatePublished - 10 Oct 2017

Bibliographical note

Publisher Copyright:
© 2017 The Authors

Keywords

  • DNA damage
  • GFP
  • differentiation
  • embryonic stem cells
  • fluorescence
  • imaging
  • live imaging
  • microscopy
  • pluripotency
  • protein dynamics

Fingerprint

Dive into the research topics of 'An Endogenously Tagged Fluorescent Fusion Protein Library in Mouse Embryonic Stem Cells'. Together they form a unique fingerprint.

Cite this