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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.

References

  1. Gingras, A.C., Gstaiger, M., Raught, B. & Aebersold, R. Nat. Rev. Mol. Cell Biol. 8, 645–654 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Van Roey, K., Dinkel, H., Weatheritt, R.J., Gibson, T.J. & Davey, N.E. Sci. Signal. 6, rs7 (2013).

    Article  PubMed  CAS  Google Scholar 

  3. Martin, K.C. & Ephrussi, A. Cell 136, 719–730 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Beck, M. et al. Mol. Syst. Biol. 7, 549 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Schwanhäusser, B. et al. Nature 473, 337–342 (2011).

    Article  PubMed  CAS  Google Scholar 

  6. Gibson, T.J. Trends Biochem. Sci. 34, 471–482 (2009).

    Article  CAS  PubMed  Google Scholar 

  7. Dumont, J.E., Dremier, S., Pirson, I. & Maenhaut, C. Am. J. Physiol. Cell Physiol. 283, C2–C28 (2002).

    Article  CAS  PubMed  Google Scholar 

  8. Nooren, I.M. & Thornton, J.M. J. Mol. Biol. 325, 991–1018 (2003).

    Article  CAS  PubMed  Google Scholar 

  9. Perkins, J.R., Diboun, I., Dessailly, B.H., Lees, J.G. & Orengo, C. Structure 18, 1233–1243 (2010).

    Article  CAS  PubMed  Google Scholar 

  10. Volonté, C., D'Ambrosi, N. & Amadio, S. Prog. Neurobiol. 86, 61–71 (2008).

    Article  PubMed  CAS  Google Scholar 

  11. Georges, A.B., Benayoun, B.A., Caburet, S. & Veitia, R.A. FASEB J. 24, 346–356 (2010).

    Article  CAS  PubMed  Google Scholar 

  12. Ettwiller, L. & Veitia, R.A. Comp. Funct. Genomics 2007, 58721 (2007).

    Article  PubMed Central  Google Scholar 

  13. Birchler, J.A. & Veitia, R.A. Proc. Natl. Acad. Sci. USA 109, 14746–14753 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Makino, T. & McLysaght, A. Proc. Natl. Acad. Sci. USA 107, 9270–9274 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Okamoto, I. et al. Nature 472, 370–374 (2011).

    Article  CAS  PubMed  Google Scholar 

  16. Cacace, A.M. et al. Mol. Cell. Biol. 19, 229–240 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Levchenko, A., Bruck, J. & Sternberg, P.W. Proc. Natl. Acad. Sci. USA 97, 5818–5823 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Calabrese, E.J. & Baldwin, L.A. Trends Pharmacol. Sci. 22, 285–291 (2001).

    Article  CAS  PubMed  Google Scholar 

  19. Vavouri, T., Semple, J.I., Garcia-Verdugo, R. & Lehner, B. Cell 138, 198–208 (2009).

    Article  CAS  PubMed  Google Scholar 

  20. Rizzo, M. A., Davidson, M. W. & Piston, D. W. Cold Spring Harb. Protoc. 2009, pdb.top64 (2009).

    Article  PubMed  Google Scholar 

  21. Lehmann, O.J. et al. Am. J. Hum. Genet. 67, 1129–1135 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Liu, Y. & Lehmann, M. Fly (Austin) 2, 92–98 (2008).

    Article  Google Scholar 

  23. Natesan, S., Rivera, V.M., Molinari, E. & Gilman, M. Nature 390, 349–350 (1997).

    Article  CAS  PubMed  Google Scholar 

  24. Peters, J.M. Nat. Rev. Mol. Cell Biol. 7, 644–656 (2006).

    Article  CAS  PubMed  Google Scholar 

  25. Nguyen, H.G., Chinnappan, D., Urano, T. & Ravid, K. Mol. Cell. Biol. 25, 4977–4992 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Stewart, S. & Fang, G. Cancer Res. 65, 8730–8735 (2005).

    Article  CAS  PubMed  Google Scholar 

  27. Dobson, C.M. Nature 426, 884–890 (2003).

    Article  CAS  PubMed  Google Scholar 

  28. Kutay, U. & Güttinger, S. Trends Cell Biol. 15, 121–124 (2005).

    Article  CAS  PubMed  Google Scholar 

  29. Diella, F. et al. Front. Biosci. 13, 6580–6603 (2008).

    Article  CAS  PubMed  Google Scholar 

  30. Hantschel, O. et al. Mol. Cell 19, 461–473 (2005).

    Article  CAS  PubMed  Google Scholar 

  31. Kadlec, J., Izaurralde, E. & Cusack, S. Nat. Struct. Mol. Biol. 11, 330–337 (2004).

    Article  CAS  PubMed  Google Scholar 

  32. Stirnimann, C.U., Ptchelkine, D., Grimm, C. & Müller, C.W. J. Mol. Biol. 400, 71–81 (2010).

    Article  CAS  PubMed  Google Scholar 

  33. Shen, D. et al. Cell Biochem. Biophys. 60, 173–185 (2011).

    Article  CAS  PubMed  Google Scholar 

  34. Roberti, M.J., Jovin, T.M. & Jares-Erijman, E. PLoS ONE 6, e23338 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Ciotta, G. et al. Methods 53, 113–119 (2011).

    Article  CAS  PubMed  Google Scholar 

  36. Poser, I. et al. Nat. Methods 5, 409–415 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Ding, L., Poser, I., Paszkowski-Rogacz, M. & Buchholz, F. Stem Cell Rev. 8, 32–42 (2012).

    Article  CAS  Google Scholar 

  38. Maliga, Z. et al. Nat. Cell Biol. 15, 325–334 (2013).

    Article  CAS  PubMed  Google Scholar 

  39. Schebelle, L. et al. Nucleic Acids Res. 38, e106 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. DeFrancesco, L. Nat. Biotechnol. 29, 681–684 (2011).

    Article  CAS  Google Scholar 

  41. Dosztányi, Z., Csizmok, V., Tompa, P. & Simon, I. Bioinformatics 21, 3433–3434 (2005).

    Article  PubMed  CAS  Google Scholar 

  42. Letunic, I., Doerks, T. & Bork, P. Nucleic Acids Res. 37, D229–D232 (2009).

    Article  CAS  PubMed  Google Scholar 

  43. Waterhouse, A.M., Procter, J.B., Martin, D.M., Clamp, M. & Barton, G.J. Bioinformatics 25, 1189–1191 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. D'Alise, A.M. et al. Mol. Cancer Ther. 7, 1140–1149 (2008).

    Article  CAS  PubMed  Google Scholar 

  45. Dong, X. et al. Nature 458, 1136–1141 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Taagepera, S. et al. Proc. Natl. Acad. Sci. USA 95, 7457–7462 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Fries, B. et al. J. Biol. Chem. 282, 4504–4515 (2007).

    Article  CAS  PubMed  Google Scholar 

  48. Giannini, A. et al. Exp. Cell Res. 295, 150–160 (2004).

    Article  CAS  PubMed  Google Scholar 

  49. Ossovskaya, V., Lim, S.T., Ota, N., Schlaepfer, D.D. & Ilic, D. FEBS Lett. 582, 2402–2406 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Roovers, K., Klein, E.A., Castagnino, P. & Assoian, R.K. Dev. Cell 5, 273–284 (2003).

    Article  CAS  PubMed  Google Scholar 

  51. Murai, N., Murakami, Y. & Matsufuji, S. J. Biol. Chem. 278, 44791–44798 (2003).

    Article  CAS  PubMed  Google Scholar 

  52. Xiao, Z., Watson, N., Rodriguez, C. & Lodish, H.F. J. Biol. Chem. 276, 39404–39410 (2001).

    Article  CAS  PubMed  Google Scholar 

  53. Begitt, A., Meyer, T., van Rossum, M. & Vinkemeier, U. Proc. Natl. Acad. Sci. USA 97, 10418–10423 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Kulisz, A. & Simon, H.G. Mol. Cell. Biol. 28, 1553–1564 (2008).

    Article  CAS  PubMed  Google Scholar 

  55. Shirley, R.L., Ford, A.S., Richards, M.R., Albertini, M. & Culbertson, M.R. Genetics 161, 1465–1482 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Turan, S. et al. J. Mol. Biol. 407, 193–221 (2011).

    Article  CAS  PubMed  Google Scholar 

  57. Osterwalder, M. et al. Nat. Methods 7, 893–895 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Kim, Y.G., Cha, J. & Chandrasegaran, S. Proc. Natl. Acad. Sci. USA 93, 1156–1160 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Zhang, F. et al. Nat. Biotechnol. 29, 149–153 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  60. Boch, J. Nat. Biotechnol. 29, 135–136 (2011).

    Article  CAS  PubMed  Google Scholar 

  61. Cong, L. et al. Science 339, 819–823 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Mali, P. et al. Science 339, 823–826 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Casola, S. Methods Mol. Biol. 667, 145–163 (2010).

    Article  CAS  PubMed  Google Scholar 

  64. Soriano, P. Nat. Genet. 21, 70–71 (1999).

    Article  CAS  PubMed  Google Scholar 

  65. Nord, A.S. et al. Nucleic Acids Res. 34, D642–D648 (2006).

    Article  CAS  PubMed  Google Scholar 

  66. Skarnes, W.C. et al. Nature 474, 337–342 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Huh, W.K. et al. Nature 425, 686–691 (2003).

    Article  CAS  PubMed  Google Scholar 

  68. Winzeler, E.A. et al. Science 285, 901–906 (1999).

    Article  CAS  PubMed  Google Scholar 

  69. Dietzl, G. et al. Nature 448, 151–156 (2007).

    Article  CAS  PubMed  Google Scholar 

Download references

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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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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