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

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 endo...

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Autores principales: Harikumar, Arigela, Edupuganti, Raghu Ram, Sorek, Matan, Azad, Gajendra Kumar, Markoulaki, Styliani, Sehnalová, Petra, Legartová, Soňa, Bártová, Eva, Farkash-Amar, Shlomit, Jaenisch, Rudolf, Alon, Uri, Meshorer, Eran
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5639459/
https://www.ncbi.nlm.nih.gov/pubmed/28966122
http://dx.doi.org/10.1016/j.stemcr.2017.08.022
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author Harikumar, Arigela
Edupuganti, Raghu Ram
Sorek, Matan
Azad, Gajendra Kumar
Markoulaki, Styliani
Sehnalová, Petra
Legartová, Soňa
Bártová, Eva
Farkash-Amar, Shlomit
Jaenisch, Rudolf
Alon, Uri
Meshorer, Eran
author_facet Harikumar, Arigela
Edupuganti, Raghu Ram
Sorek, Matan
Azad, Gajendra Kumar
Markoulaki, Styliani
Sehnalová, Petra
Legartová, Soňa
Bártová, Eva
Farkash-Amar, Shlomit
Jaenisch, Rudolf
Alon, Uri
Meshorer, Eran
author_sort Harikumar, Arigela
collection PubMed
description 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.
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spelling pubmed-56394592017-10-20 An Endogenously Tagged Fluorescent Fusion Protein Library in Mouse Embryonic Stem Cells Harikumar, Arigela Edupuganti, Raghu Ram Sorek, Matan Azad, Gajendra Kumar Markoulaki, Styliani Sehnalová, Petra Legartová, Soňa Bártová, Eva Farkash-Amar, Shlomit Jaenisch, Rudolf Alon, Uri Meshorer, Eran Stem Cell Reports Resource 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. Elsevier 2017-09-28 /pmc/articles/PMC5639459/ /pubmed/28966122 http://dx.doi.org/10.1016/j.stemcr.2017.08.022 Text en © 2017 The Authors http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Resource
Harikumar, Arigela
Edupuganti, Raghu Ram
Sorek, Matan
Azad, Gajendra Kumar
Markoulaki, Styliani
Sehnalová, Petra
Legartová, Soňa
Bártová, Eva
Farkash-Amar, Shlomit
Jaenisch, Rudolf
Alon, Uri
Meshorer, Eran
An Endogenously Tagged Fluorescent Fusion Protein Library in Mouse Embryonic Stem Cells
title An Endogenously Tagged Fluorescent Fusion Protein Library in Mouse Embryonic Stem Cells
title_full An Endogenously Tagged Fluorescent Fusion Protein Library in Mouse Embryonic Stem Cells
title_fullStr An Endogenously Tagged Fluorescent Fusion Protein Library in Mouse Embryonic Stem Cells
title_full_unstemmed An Endogenously Tagged Fluorescent Fusion Protein Library in Mouse Embryonic Stem Cells
title_short An Endogenously Tagged Fluorescent Fusion Protein Library in Mouse Embryonic Stem Cells
title_sort endogenously tagged fluorescent fusion protein library in mouse embryonic stem cells
topic Resource
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5639459/
https://www.ncbi.nlm.nih.gov/pubmed/28966122
http://dx.doi.org/10.1016/j.stemcr.2017.08.022
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