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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...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier
2017
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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. |
format | Online Article Text |
id | pubmed-5639459 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Elsevier |
record_format | MEDLINE/PubMed |
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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