TY - JOUR
T1 - An Endogenously Tagged Fluorescent Fusion Protein Library in Mouse Embryonic Stem Cells
AU - Harikumar, Arigela
AU - Edupuganti, Raghu Ram
AU - Sorek, Matan
AU - Azad, Gajendra Kumar
AU - Markoulaki, Styliani
AU - Sehnalová, Petra
AU - Legartová, Soňa
AU - Bártová, Eva
AU - Farkash-Amar, Shlomit
AU - Jaenisch, Rudolf
AU - Alon, Uri
AU - Meshorer, Eran
N1 - Publisher Copyright:
© 2017 The Authors
PY - 2017/10/10
Y1 - 2017/10/10
N2 - 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.
AB - 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.
KW - DNA damage
KW - GFP
KW - differentiation
KW - embryonic stem cells
KW - fluorescence
KW - imaging
KW - live imaging
KW - microscopy
KW - pluripotency
KW - protein dynamics
UR - http://www.scopus.com/inward/record.url?scp=85030686398&partnerID=8YFLogxK
U2 - 10.1016/j.stemcr.2017.08.022
DO - 10.1016/j.stemcr.2017.08.022
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C2 - 28966122
AN - SCOPUS:85030686398
SN - 2213-6711
VL - 9
SP - 1304
EP - 1314
JO - Stem Cell Reports
JF - Stem Cell Reports
IS - 4
ER -
