Characterization of Coelenterazine Analogs for Measurements of Luciferase Activity in Live Cells and Living Animals
In vivo imaging of bioluminescent reporters relies on expression of light-emitting enzymes, luciferases, and delivery of chemical substrates to expressing cells. Coelenterazine (CLZN) is the substrate for a group of bioluminescent enzymes obtained from marine organisms. At present, there are more th...
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| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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SAGE Publishing
2004-01-01
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| Series: | Molecular Imaging |
| Online Access: | https://doi.org/10.1162/15353500200403181 |
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| _version_ | 1832544655718219776 |
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| author | Hui Zhao Timothy C. Doyle Ronald J. Wong Yuan Cao David K. Stevenson David Piwnica-Worms Christopher H. Contag |
| author_facet | Hui Zhao Timothy C. Doyle Ronald J. Wong Yuan Cao David K. Stevenson David Piwnica-Worms Christopher H. Contag |
| author_sort | Hui Zhao |
| collection | DOAJ |
| description | In vivo imaging of bioluminescent reporters relies on expression of light-emitting enzymes, luciferases, and delivery of chemical substrates to expressing cells. Coelenterazine (CLZN) is the substrate for a group of bioluminescent enzymes obtained from marine organisms. At present, there are more than 10 commercially available CLZN analogs. To determine which analog is most suitable for activity measurements in live cells and living animals, we characterized 10 CLZN analogs using Renilla luciferase (Rluc) as the reporter enzyme. For each analog, we monitored enzyme activity, auto-oxidation, and efficiency of cellular uptake. All CLZN analogs tested showed higher auto-oxidation signals in serum than was observed in phosphate buffer or medium, mainly as a result of auto-oxidation by binding to albumin. CLZN- f , -h , and -e analogs showed 4- to 8-fold greater Rluc activity, relative to CLZN- native , in cells expressing the enzyme from a stable integrant. In studies using living mice expressing Rluc in hepatocytes, administration of CLZN- e and -native produced the highest signal. Furthermore, distinct temporal differences in signal for each analog were revealed following intravenous or intraperitoneal delivery. We conclude that the CLZN analogs that are presently available vary with respect to hRluc utilization in culture and in vivo, and that the effective use of CLZN-utilizing enzymes in living animals depends on the selection of an appropriate substrate. |
| format | Article |
| id | doaj-art-f7f35d8738784f45b7313b8aa766ac68 |
| institution | Kabale University |
| issn | 1536-0121 |
| language | English |
| publishDate | 2004-01-01 |
| publisher | SAGE Publishing |
| record_format | Article |
| series | Molecular Imaging |
| spelling | doaj-art-f7f35d8738784f45b7313b8aa766ac682025-02-03T10:08:01ZengSAGE PublishingMolecular Imaging1536-01212004-01-01310.1162/1535350020040318110.1162_15353500200403181Characterization of Coelenterazine Analogs for Measurements of Luciferase Activity in Live Cells and Living AnimalsHui Zhao0Timothy C. Doyle1Ronald J. Wong2Yuan Cao3David K. Stevenson4David Piwnica-Worms5Christopher H. Contag6Stanford University School of MedicineStanford University School of MedicineStanford University School of MedicineStanford University School of MedicineStanford University School of MedicineMallinckrodt Institute of Radiology and Washington University School of MedicineStanford University School of MedicineIn vivo imaging of bioluminescent reporters relies on expression of light-emitting enzymes, luciferases, and delivery of chemical substrates to expressing cells. Coelenterazine (CLZN) is the substrate for a group of bioluminescent enzymes obtained from marine organisms. At present, there are more than 10 commercially available CLZN analogs. To determine which analog is most suitable for activity measurements in live cells and living animals, we characterized 10 CLZN analogs using Renilla luciferase (Rluc) as the reporter enzyme. For each analog, we monitored enzyme activity, auto-oxidation, and efficiency of cellular uptake. All CLZN analogs tested showed higher auto-oxidation signals in serum than was observed in phosphate buffer or medium, mainly as a result of auto-oxidation by binding to albumin. CLZN- f , -h , and -e analogs showed 4- to 8-fold greater Rluc activity, relative to CLZN- native , in cells expressing the enzyme from a stable integrant. In studies using living mice expressing Rluc in hepatocytes, administration of CLZN- e and -native produced the highest signal. Furthermore, distinct temporal differences in signal for each analog were revealed following intravenous or intraperitoneal delivery. We conclude that the CLZN analogs that are presently available vary with respect to hRluc utilization in culture and in vivo, and that the effective use of CLZN-utilizing enzymes in living animals depends on the selection of an appropriate substrate.https://doi.org/10.1162/15353500200403181 |
| spellingShingle | Hui Zhao Timothy C. Doyle Ronald J. Wong Yuan Cao David K. Stevenson David Piwnica-Worms Christopher H. Contag Characterization of Coelenterazine Analogs for Measurements of Luciferase Activity in Live Cells and Living Animals Molecular Imaging |
| title | Characterization of Coelenterazine Analogs for Measurements of Luciferase Activity in Live Cells and Living Animals |
| title_full | Characterization of Coelenterazine Analogs for Measurements of Luciferase Activity in Live Cells and Living Animals |
| title_fullStr | Characterization of Coelenterazine Analogs for Measurements of Luciferase Activity in Live Cells and Living Animals |
| title_full_unstemmed | Characterization of Coelenterazine Analogs for Measurements of Luciferase Activity in Live Cells and Living Animals |
| title_short | Characterization of Coelenterazine Analogs for Measurements of Luciferase Activity in Live Cells and Living Animals |
| title_sort | characterization of coelenterazine analogs for measurements of luciferase activity in live cells and living animals |
| url | https://doi.org/10.1162/15353500200403181 |
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