Cytotechnology

, Volume 68, Issue 1, pp 19–24 | Cite as

Derivation of a novel G2 reporter system

  • Elizabeth Fidalgo da Silva
  • Sabrina Botsford
  • Lisa A. Porter
Technical Note

Abstract

Progression through G2 phase of the cell cycle is a technically difficult area of cell biology to study due to the lack of physical markers specific to this phase. The FUCCI system uses the biology of the cell cycle to drive fluorescence in select phases of the cell cycle. Similarly, a commercially available system has used a fluorescent analog of the Cyclin B1 protein to visualize cells from late S phase to the metaphase–anaphase transition. We have modified these systems to use the promoter and destruction box elements of Cyclin B1 to drive a cyan fluorescent protein. We demonstrate here that this is a useful tool for measuring the length of G2 phase without perturbing any aspect of cell cycle progression.

Keywords

Cell cycle G2 phase Mitosis DNA synthesis Cyclin B1 

Notes

Acknowledgments

We thank Bob Hodge and Jiamila Maimaiti for technical assistance. We also thank Dorota Lubanska and Kaitlyn Matthews for feedback on this work. S.B. acknowledges support from the Natural Science and Engineering Research Council of Canada (NSERC) Undergraduate Student Research Assistantship Program (USRA). This work was supported by an NSERC grant (#312014-2009).

Conflict of interest

The authors have no conflicts to declare.

References

  1. Barnes EA, Kong M, Ollendorff V, Donoghue DJ (2001) Patched1 interacts with cyclin B1 to regulate cell cycle progression. EMBO J 20:2214–2223CrossRefGoogle Scholar
  2. Berridge MV, Herst PM, Tan AS (2005) Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol Annu Rev 11:127–152CrossRefGoogle Scholar
  3. Bostock CJ, Prescott DM, Kirkpatrick JB (1971) An evaluation of the double thymidine block for synchronizing mammalian cells at the G1-S border. Exp Cell Res 68:163–168CrossRefGoogle Scholar
  4. Cogswell JP, Godlevski MM, Bonham M, Bisi J, Babiss L (1995) Upstream stimulatory factor regulates expression of the cell cycle-dependent cyclin B1 gene promoter. Mol Cell Biol 15:2782–2790CrossRefGoogle Scholar
  5. Fidalgo da Silva E, Ansari SB, Maimaiti J, Barnes EA, Kong-Beltran M, Donoghue DJ, Porter LA (2011) The tumor suppressor tuberin regulates mitotic onset through the cellular localization of cyclin B1. Cell Cycle 10:3129–3139CrossRefGoogle Scholar
  6. Fidalgo da Silva E, Martinello KB, Hanna MA, Porter LA (2013) Interactions between Tuberin and Cyclin B1 function as a G2/M growth factor sensor. EMBO Reports Submitted (EMBOR-2013-38151V1)Google Scholar
  7. Hagting A, Karlsson C, Clute P, Jackman M, Pines J (1998) MPF localization is controlled by nuclear export. EMBO J 17:4127–4138CrossRefGoogle Scholar
  8. King RW, Deshaies RJ, Peters JM, Kirschner MW (1996a) How proteolysis drives the cell cycle. Science 274:1652–1659CrossRefGoogle Scholar
  9. King RW, Glotzer M, Kirschner MW (1996b) Mutagenic analysis of the destruction signal of mitotic cyclins and structural characterization of ubiquitinated intermediates. Mol Biol Cell 7:1343–1357CrossRefGoogle Scholar
  10. Lim S, Kaldis P (2013) Cdks, cyclins and CKIs: roles beyond cell cycle regulation. Development 140:3079–3093CrossRefGoogle Scholar
  11. Meyn MA 3rd, Melloy PG, Li J, Holloway SL (2002) The destruction box of the cyclin Clb2 binds the anaphase-promoting complex/cyclosome subunit Cdc23. Arch Biochem Biophys 407:189–195CrossRefGoogle Scholar
  12. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63CrossRefGoogle Scholar
  13. Passmore LA, McCormack EA, Au SW, Paul A, Willison KR, Harper JW, Barford D (2003) Doc1 mediates the activity of the anaphase-promoting complex by contributing to substrate recognition. EMBO J 22:786–796CrossRefGoogle Scholar
  14. Piaggio G, Farina A, Perrotti D, Manni I, Fuschi P, Sacchi A, Gaetano C (1995) Structure and growth-dependent regulation of the human cyclin B1 promoter. Exp Cell Res 216:396–402CrossRefGoogle Scholar
  15. Pines J, Hunter T (1989) Isolation of a human cyclin cDNA: evidence for cyclin mRNA and protein regulation in the cell cycle and for interaction with p34cdc2. Cell 58:833–846CrossRefGoogle Scholar
  16. Qin JY, Zhang L, Clift KL, Hulur I, Xiang AP, Ren BZ, Lahn BT (2010) Systematic comparison of constitutive promoters and the doxycycline-inducible promote. PLoS One 5:e10611CrossRefGoogle Scholar
  17. Reed SE, Staley EM, Mayginnes JP, Pintel DJ, Tullis GE (2006) Transfection of mammalian cells using linear polyethylenimine is a simple and effective means of producing recombinant adeno-associated virus vectors. J Virol Methods 138:85–98CrossRefGoogle Scholar
  18. Sakaue-Sawano A, Kurokawa H, Morimura T, Hanyu A, Hama H, Osawa H, Kashiwagi S, Fukami K, Miyata T, Miyoshi H, Imamura T, Ogawa M, Masai H, Miyawaki A (2008) Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell 132:487–498CrossRefGoogle Scholar
  19. Yamano H, Gannon J, Mahbubani H, Hunt T (2004) Cell cycle-regulated recognition of the destruction box of cyclin B by the APC/C in Xenopus egg extracts. Mol Cell 13:137–147CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Elizabeth Fidalgo da Silva
    • 1
  • Sabrina Botsford
    • 1
  • Lisa A. Porter
    • 1
  1. 1.Department of Biological SciencesUniversity of WindsorWindsorCanada

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