3 years ago

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

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 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.

Graphical abstract

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Teaser

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.

Publisher URL: www.sciencedirect.com/science

DOI: S221367111730379X

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