Optimization of enzyme - Substrate pairing for bioluminescence imaging of gene transfer using Renilla and Gaussia luciferases

Takahiro Kimura, Kei Hiraoka, Noriyuki Kasahara, Christopher R. Logg

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

Background: Bioluminescence imaging (BLI) permits the non-invasive quantification and localization of transduction and expression by gene transfer vectors. The tendency of tissue to attenuate light in the optical region, however, limits the sensitivity of BLI. Improvements in light output from bioluminescent reporter systems would allow the detection of lower levels of expression, smaller numbers of cells and expression from deeper and more attenuating tissues within an animal. Methods: With the goal of identifying substrates that allow improved sensitivity with Renilla luciferase (RLuc) and Gaussia luciferase (GLuc) reporter genes, we evaluated native coelenterazine and three of its most promising derivatives in BLI of cultured cells transduced with retroviral vectors encoding these reporters. Of the eight enzyme - substrate pairs tested, the two that performed best were further evaluated in mice to compare their effectiveness for imaging vector-modified cells in live animals. Results: In cell culture, we observed striking differences in luminescence levels from the various enzyme-substrate combinations and found that the two luciferases exhibited markedly distinct abilities to generate light with the substrates. The most effective pairs were RLuc with the synthetic coelenterazine derivative ViviRen, and GLuc with native coelenterazine. In animals, these two pairs allowed similar detection sensitivities, which were eight- to 15-fold higher than that of the prototypical RLuc-native coelenterazine combination. Conclusions: Substrate selection can dramatically influence the detection sensitivity of RLuc and GLuc and appropriate choice of substrate can greatly improve the performance of reporter genes encoding these enzymes for monitoring gene transfer by BLI.

Original languageEnglish (US)
Pages (from-to)528-537
Number of pages10
JournalJournal of Gene Medicine
Volume12
Issue number6
DOIs
StatePublished - Jun 2010
Externally publishedYes

Fingerprint

Renilla Luciferases
Luciferases
Enzymes
Reporter Genes
Light
Genes
Luminescence
Cultured Cells
Cell Culture Techniques
Cell Count
Gene Expression
coelenterazine

Keywords

  • Bioluminescence
  • Coelenterazine
  • Imaging
  • Luciferase
  • Reporter gene
  • Retrovirus

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology
  • Molecular Medicine
  • Genetics(clinical)
  • Drug Discovery
  • Medicine(all)

Cite this

Optimization of enzyme - Substrate pairing for bioluminescence imaging of gene transfer using Renilla and Gaussia luciferases. / Kimura, Takahiro; Hiraoka, Kei; Kasahara, Noriyuki; Logg, Christopher R.

In: Journal of Gene Medicine, Vol. 12, No. 6, 06.2010, p. 528-537.

Research output: Contribution to journalArticle

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