Assaying NanoLuc Luciferase Activity from mRNA-Injected Xenopus Embryos

  • Michael D. SheetsEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1920)


The earliest steps of animal development depend upon posttranscriptional events that drive the embryonic cell cycle and guide cell fate decisions. The analysis of post-transcriptional regulatory events has relied upon the use of chimeric reporter mRNAs that encode firefly luciferase fused to potential regulatory sequences. A new and more sensitive luciferase developed recently called NanoLuc has the potential to improve reporter studies and provide new insights into the regulation of embryonic processes. Here I describe how to create and analyze reporter mRNAs encoding NanoLuc luciferase using extracts from microinjected Xenopus embryos.

Key words

Xenopus embryos mRNA microinjection NanoLuc Luciferase 



Work in the Sheets lab was supported by NIH grants (R21HD076828, R01HD091921).


  1. 1.
    Sheets MD, Fox CA, Hunt T, Vande Woude G, Wickens M (1994) The 3′-untranslated regions of c-mos and cyclin mRNAs stimulate translation by regulating cytoplasmic polyadenylation. Genes Dev 8:926–938CrossRefGoogle Scholar
  2. 2.
    Arnone MI, Dmochowski IJ, Gache C (2004) Using reporter genes to study cis-regulatory elements. Methods Cell Biol 74:621–652CrossRefGoogle Scholar
  3. 3.
    Miraglia LJ, King FJ, Damoiseaux R (2011) Seeing the light: luminescent reporter gene assays. Comb Chem High Throughput Screen 14:648–657CrossRefGoogle Scholar
  4. 4.
    Gallie DR (1991) The cap and poly(A) tail function synergistically to regulate mRNA translational efficiency. Genes Dev 5:2108–2116CrossRefGoogle Scholar
  5. 5.
    Rizzo MA, Davidson MW, Piston DW (2009) Fluorescent protein tracking and detection: applications using fluorescent proteins in living cells. Cold Spring Harb Protoc 2009:pdb top64CrossRefGoogle Scholar
  6. 6.
    Giepmans BN, Adams SR, Ellisman MH, Tsien RY (2006) The fluorescent toolbox for assessing protein location and function. Science 312:217–224CrossRefGoogle Scholar
  7. 7.
    de Wet JR, Wood KV, Helinski DR, DeLuca M (1985) Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli. Proc Natl Acad Sci U S A 82:7870–7873CrossRefGoogle Scholar
  8. 8.
    Hooper CE, Ansorge RE, Rushbrooke JG (1994) Low-light imaging technology in the life sciences. J Biolumin Chemilumin 9:113–122CrossRefGoogle Scholar
  9. 9.
    Hall MP, Unch J, Binkowski BF, Valley MP, Braeden LB, Wood MG et al (2012) Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol 7:1848–1857CrossRefGoogle Scholar
  10. 10.
    England CG, Ehlerding EB, Cai W (2016) NanoLuc: a small luciferase is brightening up the field of bioluminescence. Bioconjug Chem 27:1175–1187CrossRefGoogle Scholar
  11. 11.
    Nilsen TW, Rio DC (2012) In vitro transcription of labeled RNA: synthesis, capping, and substitution. Cold Spring Harb Protoc 2012:1181–1186PubMedGoogle Scholar
  12. 12.
    Rio DC (2015) Denaturation and electrophoresis of RNA with glyoxal. Cold Spring Harb Protoc 2015:223–226PubMedGoogle Scholar
  13. 13.
    Rio DC (2015) Denaturation and electrophoresis of RNA with formaldehyde. Cold Spring Harb Protoc 2015:219–222PubMedGoogle Scholar
  14. 14.
    Park S, Blaser S, Marchal MA, Houston DW, Sheets MD (2016) A gradient of maternal Bicaudal-C controls vertebrate embryogenesis via translational repression of mRNAs encoding cell fate regulators. Development 143:864–871CrossRefGoogle Scholar
  15. 15.
    Zhang Y, Cooke A, Park s, Dewey CN, Wickens M, Sheets MD (2013) Bicaudal-C spatially controls translation of vertebrate maternal mRNAs. RNA 19:1575–1582CrossRefGoogle Scholar
  16. 16.
    Zhang Y, Forinash KD, McGivern J, Fritz B, Dorey K, Sheets MD (2009) Spatially restricted translation of the xCR1 mRNA in Xenopus embryos. Mol Cell Biol 29:3791–3802CrossRefGoogle Scholar
  17. 17.
    Sive HL, Grainger RM, Harland RM (2010) Microinjection of Xenopus embryos. Cold Spring Harb Protoc 2010:pdb ip81CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biomolecular ChemistrySchool of Medicine and Public Health, University of Wisconsin-MadisonMadisonUSA

Personalised recommendations