Optimization of enzyme-substrate pairing for bioluminescence imaging of gene transfer using Renilla and Gaussia luciferases.
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.
Department of Medicine, University of California, Los Angeles, CA, USA.
Takahiro
T
Kimura
Kei
K
Hiraoka
Noriyuki
N
Kasahara
Christopher R
CR
Logg
D000818:Animals; D002460:Cell Line; D033342:Copepoda / Q000201:enzymology*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D006801:Humans; D007093:Imidazoles / Q000737:chemistry / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D049408:Luminescent Agents / Q000378:metabolism; D008163:Luminescent Measurements / Q000295:instrumentation / Q000379:methods*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008819:Mice, Nude; D011719:Pyrazines / Q000737:chemistry / Q000378:metabolism; D048790:Renilla / Q000201:enzymology*
D007093:Imidazoles; D049408:Luminescent Agents; D008164:Luminescent Proteins; D011719:Pyrazines; C017144:coelenterazine; D008156:Luciferases