School of Medicine and Health Sciences Poster Presentations

A Synthetic Luciferin Improves In Vivo Bioluminescence Imaging of Gene Expression in Cardiovascular Brain Regions of Mice

Poster Number

123

Document Type

Poster

Publication Date

3-2016

Abstract

Bioluminescence imaging is a powerful tool for in vivo investigation of biological processes. Incorporation of firefly luciferase into whole animals or organ specific areas, combined with exogenous administration of the substrate D-Luciferin, results in light production that can be captured with a charge-coupled device camera. We have demonstrated the utility of in vivo bioluminescence imaging to spatiotemporally monitor gene expression and transcription factor activation in individual cardiovascular brain nuclei during the development of cardiovascular disease. However, D-Luciferin uptake into the brain is low, which may limit the sensitivity of bioluminescence imaging, particularly when considering small changes in gene expression in single central nervous system areas. Therefore, approaches that improve the sensitivity of in vivo bioluminescence imaging are warranted. Here, we tested the hypothesis that a synthetic luciferase substrate, cyclic alkylaminoluciferin (CycLuc1), would be superior to D-Luciferin for in vivo monitoring of gene expression in cardiovascular brain regions. Male C57B1/6 mice (n=4) underwent targeted delivery of an adenovirus encoding the luciferase gene (luc) downstream of the CMV promoter to the subfornical organ, a circumventricular brain region that is critical in the control of the cardiovascular system. Following gene transfer and recovery, D-Luciferin or CycLuc1 were administered in a randomized fashion on 2 separate days and bioluminescence imaging was performed using an IVIS Lumina K system. The substrate dose (150 mg/kg) was similar between conditions and was chosen based on the typical use of this concentration for in vivo imaging with D-Luciferin. Administration of D-Luciferin revealed a bioluminescent signal from the subfornical organ of 3.2±1.2 x 105 photons/s at 10 minutes after substrate administration. In contrast, in the same animals at an equivalent concentration (figure), CycLuc1 injection was associated with a more intense light emission (7.7±2.6 x 105; p=0.06 vs. D-Luciferin) that was approximately 3-fold greater than that found with D-Luciferin (2.9±0.9 fold D-Luciferin). Similarly, at 20 minutes post substrate administration CycLuc1 provided a 3.3±1.1 fold higher bioluminescent signal than D-Luciferin (2.9±1.2 vs. 7.6±2.6 x 105 photons/s; D-Luciferin vs. CycLuc1; p=0.05). These preliminary findings demonstrate that replacing standard D-Luciferin with the synthetic luciferin CycLuc1 improves the sensitivity of bioluminescent detection from individual central nervous system cardiovascular control areas.

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This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

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Presented at: GW Research Days 2016

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A Synthetic Luciferin Improves In Vivo Bioluminescence Imaging of Gene Expression in Cardiovascular Brain Regions of Mice

Bioluminescence imaging is a powerful tool for in vivo investigation of biological processes. Incorporation of firefly luciferase into whole animals or organ specific areas, combined with exogenous administration of the substrate D-Luciferin, results in light production that can be captured with a charge-coupled device camera. We have demonstrated the utility of in vivo bioluminescence imaging to spatiotemporally monitor gene expression and transcription factor activation in individual cardiovascular brain nuclei during the development of cardiovascular disease. However, D-Luciferin uptake into the brain is low, which may limit the sensitivity of bioluminescence imaging, particularly when considering small changes in gene expression in single central nervous system areas. Therefore, approaches that improve the sensitivity of in vivo bioluminescence imaging are warranted. Here, we tested the hypothesis that a synthetic luciferase substrate, cyclic alkylaminoluciferin (CycLuc1), would be superior to D-Luciferin for in vivo monitoring of gene expression in cardiovascular brain regions. Male C57B1/6 mice (n=4) underwent targeted delivery of an adenovirus encoding the luciferase gene (luc) downstream of the CMV promoter to the subfornical organ, a circumventricular brain region that is critical in the control of the cardiovascular system. Following gene transfer and recovery, D-Luciferin or CycLuc1 were administered in a randomized fashion on 2 separate days and bioluminescence imaging was performed using an IVIS Lumina K system. The substrate dose (150 mg/kg) was similar between conditions and was chosen based on the typical use of this concentration for in vivo imaging with D-Luciferin. Administration of D-Luciferin revealed a bioluminescent signal from the subfornical organ of 3.2±1.2 x 105 photons/s at 10 minutes after substrate administration. In contrast, in the same animals at an equivalent concentration (figure), CycLuc1 injection was associated with a more intense light emission (7.7±2.6 x 105; p=0.06 vs. D-Luciferin) that was approximately 3-fold greater than that found with D-Luciferin (2.9±0.9 fold D-Luciferin). Similarly, at 20 minutes post substrate administration CycLuc1 provided a 3.3±1.1 fold higher bioluminescent signal than D-Luciferin (2.9±1.2 vs. 7.6±2.6 x 105 photons/s; D-Luciferin vs. CycLuc1; p=0.05). These preliminary findings demonstrate that replacing standard D-Luciferin with the synthetic luciferin CycLuc1 improves the sensitivity of bioluminescent detection from individual central nervous system cardiovascular control areas.