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

doi:10.1016/j.stemcr.2017.08.022

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

Department of Genetics

Research output: Contribution to journal › Article › peer-review

15 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 language	American English
Pages (from-to)	1304-1314
Number of pages	11
Journal	Stem Cell Reports
Volume	9
Issue number	4
DOIs	https://doi.org/10.1016/j.stemcr.2017.08.022
State	Published - 10 Oct 2017

Bibliographical note

Funding Information:
This work was supported by the Israel Science Foundation ( ISF 1140/2017 to E.M. and ISF 1349/15 to U.A.); the European Research Council (to E.M.); the European Union's Horizon 2020 research and innovation program FET-OPEN ( CellViewer , no. 686637 ); and NIH grants HD045022 , R37-CA084198 , and R01 NS088538-01 (to R.J.). E.M. is the incumbent of the Arthur Gutterman Professor Chair. U.A. is the incumbent of the Abisch-Frenkel Professor Chair. R.R.E. and A.H. were supported by the Marie Curie Nucleosome4D network ; R.R.E. was supported by a post-doctoral fellowship from the Lady Davis Foundation . The authors thank Dan Lehman for help with single-cell sorting, as well as Bill Breuer, Tamar Danon, Mayan Sheiba, David Gokhman, Naveh Evantal, and Lilya Verchovsky for technical assistance. We dedicate this paper to the memory of Lilya Verchovsky.

Publisher Copyright:
© 2017 The Authors

Keywords

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

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10.1016/j.stemcr.2017.08.022

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@article{0009b099ae1249e5af42754fad8a85ae,

title = "An Endogenously Tagged Fluorescent Fusion Protein Library in Mouse Embryonic Stem Cells",

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.",

keywords = "DNA damage, GFP, differentiation, embryonic stem cells, fluorescence, imaging, live imaging, microscopy, pluripotency, protein dynamics",

author = "Arigela Harikumar and Edupuganti, {Raghu Ram} and Matan Sorek and Azad, {Gajendra Kumar} and Styliani Markoulaki and Petra Sehnalov{\'a} and So{\v n}a Legartov{\'a} and Eva B{\'a}rtov{\'a} and Shlomit Farkash-Amar and Rudolf Jaenisch and Uri Alon and Eran Meshorer",

note = "Funding Information: This work was supported by the Israel Science Foundation ( ISF 1140/2017 to E.M. and ISF 1349/15 to U.A.); the European Research Council (to E.M.); the European Union's Horizon 2020 research and innovation program FET-OPEN ( CellViewer , no. 686637 ); and NIH grants HD045022 , R37-CA084198 , and R01 NS088538-01 (to R.J.). E.M. is the incumbent of the Arthur Gutterman Professor Chair. U.A. is the incumbent of the Abisch-Frenkel Professor Chair. R.R.E. and A.H. were supported by the Marie Curie Nucleosome4D network ; R.R.E. was supported by a post-doctoral fellowship from the Lady Davis Foundation . The authors thank Dan Lehman for help with single-cell sorting, as well as Bill Breuer, Tamar Danon, Mayan Sheiba, David Gokhman, Naveh Evantal, and Lilya Verchovsky for technical assistance. We dedicate this paper to the memory of Lilya Verchovsky. Publisher Copyright: {\textcopyright} 2017 The Authors",

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doi = "10.1016/j.stemcr.2017.08.022",

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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

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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.

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KW - GFP

KW - differentiation

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KW - fluorescence

KW - imaging

KW - live imaging

KW - microscopy

KW - pluripotency

KW - protein dynamics

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

Abstract

Bibliographical note

Keywords

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