期刊
JOURNAL OF GENE MEDICINE
卷 12, 期 6, 页码 528-537出版社
WILEY
DOI: 10.1002/jgm.1463
关键词
bioluminescence; coelenterazine; imaging; luciferase; reporter gene; retrovirus
资金
- NCI [R01 CA105171, R01 CA121258, R25 CA098010, P50 CA086306, 2U24 CA092865, P30 CA016042]
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. Copyright (C) 2010 John Wiley & Sons, Ltd.
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