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The transience of transient overexpression

Much of what is known about mammalian cell regulation has been achieved with the aid of transiently transfected cells. However, overexpression can violate balanced gene dosage, affecting protein folding, complex assembly and downstream regulation. To avoid these problems, genome engineering technologies now enable the generation of stable cell lines expressing modified proteins at (almost) native levels.

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Figure 1: Illustration of how varying a scaffold concentration can result in a bell-shaped curve of signaling protein activity.
Figure 2: Experimentally investigated candidates for the anaphase destruction motif in aurora B.
Figure 3: Structural context of one true and three postulated NESs in human proteins.
Figure 4: Examples of discretely localized cellular proteins in engineered cell lines stably expressing GFP-tagged transgenes.

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Acknowledgements

We thank many colleagues at the European Molecular Biology Laboratory, Monod Institute and two research consortia, the German National Genome Research Network–funded DiGtoP and European Union–funded SyBoSS—which are focused on developing and applying genome engineered cell lines—for useful discussions. We apologize for not citing many important references because of space limitations. M.S. is funded by DiGtoP. R.A.V. is supported by Centre National de la Recherche Scientifique, La Ligue contre le Cancer (Comité de Paris), l'Université Paris Diderot–Paris 7 and Institut Universitaire de France. Special thanks to I. Poser, M. Augsburg and A. Nitzsche (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden) for providing the images of stably engineered cell lines.

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Correspondence to Toby J Gibson or Reiner A Veitia.

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Gibson, T., Seiler, M. & Veitia, R. The transience of transient overexpression. Nat Methods 10, 715–721 (2013). https://doi.org/10.1038/nmeth.2534

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