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Central European Journal of Biology

, Volume 8, Issue 5, pp 423–431 | Cite as

The Luc2 gene enhances reliability of bicistronic assays

  • Tomáš MašekEmail author
  • Václav Vopalenský
  • Martin Pospíšek
Communication
  • 390 Downloads

Abstract

Luciferases are prominent reporters in molecular and cellular biology investigations including miRNA target studies and the determination of Internal Ribosome Entry Site (IRES) activities in bicistronic assays. A majority of the current bicistronic vectors contain a firefly luciferase reporter as the second cistron. One reason for this is the presence of cryptic transcription start sites inside the luciferase gene. We present here an experimental evaluation of the cryptic transcription within the latest version of the firefly luciferase gene, luc2. Using flow cytometric analysis, we observed a negligible amount of cryptic transcriptional activity that was only slightly above the background of untransfected cells. Nevertheless, quantitative reverse transcription PCR experiments revealed a six-to-nine-fold gradual increase of transcription along the coding region of the gene. The level of cryptic transcription from the coding region of the improved luc2 firefly luciferase gene is significantly lower when compared to the luc+ gene. In summary, the luc2 better fulfills the requirements of bicistronic assays than the previous luc+ version. The observed low cryptic transcription activity in luc2 could be limiting only in cases where weak IRESs are studied.

Keywords

Luciferase Cryptic transcription Reporter gene Bicistronic vector IRES 

Abbreviations

Ct

threshold cycle

IRES

Internal ribosome entry site

NMD

nonsense-mediated decay

qRT-PCR

quantitative reverse transcription PCR

sORF

short ORF

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Copyright information

© Versita Warsaw and Springer-Verlag Wien 2013

Authors and Affiliations

  • Tomáš Mašek
    • 1
    Email author
  • Václav Vopalenský
    • 1
  • Martin Pospíšek
    • 1
  1. 1.Department of Genetics and Microbiology, Faculty of ScienceCharles University in PraguePragueCzech Republic

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