Dataset Information

ATP-Independent Bioluminescent Reporter Variants To Improve in Vivo Imaging.

ABSTRACT: Coelenterazine (CTZ)-utilizing marine luciferases and their derivatives have attracted significant attention because of their ATP-independency, fast enzymatic turnover, and high bioluminescence brightness. However, marine luciferases typically emit blue photons and their substrates, including CTZ and the recently developed diphenylterazine (DTZ), have poor water solubility, hindering their in vivo applications. Herein, we report a family of pyridyl CTZ and DTZ analogs that exhibit spectrally shifted emission and improved water solubility. Through directed evolution, we engineered a LumiLuc luciferase with broad substrate specificity. In the presence of corresponding pyridyl substrates (i.e., pyCTZ, 6pyDTZ, or 8pyDTZ), LumiLuc generates highly bright blue, teal, or yellow bioluminescence. We compared our LumiLuc-8pyDTZ pair with several benchmark reporters in a tumor xenograft mouse model. Our new pair, which does not need organic cosolvents for in vivo administration, surpasses other reporters by detecting early tumors. We further fused LumiLuc to a red fluorescent protein, resulting in a LumiScarlet reporter with further red-shifted emission and enhanced tissue penetration. LumiScarlet-8pyDTZ was comparable to Akaluc-AkaLumine, the brightest ATP-dependent luciferase-luciferin pair, for detecting cells in deep tissues of mice. In summary, we have engineered a new family of ATP-independent bioluminescent reporters, which will have broad applications because of their ATP-independency, excellent biocompatibility, and superior in vivo sensitivity.

SUBMITTER: Yeh HW

PROVIDER: S-EPMC6528180 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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ATP-Independent Bioluminescent Reporter Variants To Improve in Vivo Imaging.

Yeh Hsien-Wei HW Xiong Ying Y Wu Tianchen T Chen Minghai M Ji Ao A Li Xinyu X Ai Hui-Wang HW

ACS chemical biology 20190417 5

Coelenterazine (CTZ)-utilizing marine luciferases and their derivatives have attracted significant attention because of their ATP-independency, fast enzymatic turnover, and high bioluminescence brightness. However, marine luciferases typically emit blue photons and their substrates, including CTZ and the recently developed diphenylterazine (DTZ), have poor water solubility, hindering their in vivo applications. Herein, we report a family of pyridyl CTZ and DTZ analogs that exhibit spectrally shi ...[more]

PMID: 30969754

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Project description:Mouse models are common tools for examining post-traumatic osteoarthritis (OA), which involves cartilage deterioration following injury or stress. One challenge to current mouse models is longitudinal monitoring of the cartilage deterioration in vivo in the same mouse during an experiment. The objective of this study was to assess the feasibility for using a novel transgenic mouse for non-invasive quantification of cartilage. Chondrocytes are defined by expression of the matrix protein aggrecan, and we developed a novel mouse containing a reporter luciferase cassette under the inducible control of the endogenous aggrecan promoter. We generated these mice by crossing a Cre-dependent luciferase reporter allele with an aggrecan creERT2 knockin allele. The advantage of this design is that the targeted knockin retains the intact endogenous aggrecan locus and expresses the tamoxifen-inducible CreERT2 protein from a second IRES-driven open reading frame. These mice display bioluminescence in the joints, tail, and trachea, consistent with patterns of aggrecan expression. To evaluate this mouse as a technology for non-invasive quantification of cartilage loss, we characterized the relationship between loss of bioluminescence and loss of cartilage after induction with (i) ex vivo collagenase digestion, (ii) an in vivo OA model utilizing treadmill running, and (iii) age. Ex vivo experiments revealed that collagenase digestion of the femur reduced both luciferase signal intensity and pixel area, demonstrating a link between cartilage degradation and bioluminescence. In an in vivo model of experimental OA, we found decreased bioluminescent signal and pixel area, which correlated with pathological disease. We detected a decrease in both bioluminescent signal intensity and area with natural aging from 2 to 13 months of age. These results indicate that the bioluminescent signal from this mouse may be used as a non-invasive quantitative measure of cartilage. Future studies may use this reporter mouse to advance basic and preclinical studies of murine experimental OA with applications in synovial joint biology, disease pathogenesis, and drug delivery.

Project description:BackgroundVascular endothelial growth factor receptor-2 (VEGFR2) plays a pivotal role in angiogenesis by eliciting vascular endothelial cell growth when bound to VEGF, a powerful pro-angiogenic ligand. While Vegf and Vegfr2 are expressed throughout gestation, the latter third of gestation in mice is characterized by a marked increase in fetoplacental angiogenesis. Thus, the objective of this study was to determine the feasibility of monitoring fetoplacental Vegfr2 gene activity non-invasively using a Vegfr2-luc reporter transgenic mouse and bioluminescent imaging.MethodsImaging parameters were optimized using two wild-type (WT) females, bearing Vegfr2-luc fetuses. Then, seven WT females, bred to Vegfr2-luc males, were imaged from gestational day (GD) 12 to 18 to determine the usefulness of the Vegfr2-luc mouse as a model for studying fetoplacental Vegfr2 activity during pregnancy. Semi-quantitative RT-PCR of Vegfr2 was also performed on whole fetoplacental units during this time. Additionally, resultant neonates were imaged at postnatal day (PND) 7, 14 and 21 to monitor Vegfr2 activity during post-natal development.ResultsFetoplacental Vegfr2 gene activity was detected as light emissions beginning on GD 12 of gestation and increased throughout the imaging period (P < 0.05), and this paralleled the Vegfr2 mRNA data obtained from RT-PCR analysis. A decline in fetoplacental light emissions was associated with a poor pregnancy outcome in one pregnancy, indicating that this approach has potential use for studies monitoring pregnancy well being. Additionally, neonatal Vegfr2 activity was detected at PND 7, 14 and 21 but declined with time (P < 0.0001).ConclusionsIn utero fetoplacental Vegfr2 gene activity was monitored longitudinally in a quantitative manner using a luciferase reporter gene and bioluminescent imaging during the latter third of gestation. This study demonstrates the feasibility of using the Vegfr2-luc mouse to monitor late gestation fetoplacental angiogenic activity under normal and experimental conditions. Additionally, neonatal Vegfr2 gene activity was monitored for three weeks postpartum, allowing continuous monitoring of Vegfr2 activity during the latter third of gestation and postnatal development within the same animals.

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ATP-Independent Bioluminescent Reporter Variants To Improve in Vivo Imaging.

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ATP-Independent Bioluminescent Reporter Variants To Improve in Vivo Imaging.

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