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...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
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Elsevier
2018
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| Online Access: | http://hdl.handle.net/1721.1/116603 |
| _version_ | 1811098447601729536 |
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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 |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology 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 | MIT |
| 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. Keywords: embryonic stem cells; imaging; live imaging; fluorescence; differentiation; pluripotency; GFP; microscopy; DNA damage; protein dynamics |
| first_indexed | 2024-09-23T17:15:08Z |
| format | Article |
| id | mit-1721.1/116603 |
| institution | Massachusetts Institute of Technology |
| last_indexed | 2024-09-23T17:15:08Z |
| publishDate | 2018 |
| publisher | Elsevier |
| record_format | dspace |
| spelling | mit-1721.1/1166032022-10-03T11:25:15Z 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 Massachusetts Institute of Technology. Department of Biology Jaenisch, Rudolf 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 National Institutes of Health (U.S.) (Grant HD045022) National Institutes of Health (U.S.) (Grant R37-CA084198) National Institutes of Health (U.S.) (Grant R01NS088538-01) 2018-06-26T13:48:09Z 2018-06-26T13:48:09Z 2017-09 2017-08 2018-06-25T19:54:36Z Article http://purl.org/eprint/type/JournalArticle 2213-6711 http://hdl.handle.net/1721.1/116603 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 http://dx.doi.org/10.1016/J.STEMCR.2017.08.022 Stem Cell Reports Creative Commons Attribution-NonCommercial-NoDerivs License http://creativecommons.org/licenses/by-nc-nd/4.0/ application/pdf Elsevier Elsevier |
| spellingShingle | 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 |
| url | http://hdl.handle.net/1721.1/116603 |
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