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The signaling complex around voltage-gated sodium (Nav) channels includes accessory proteins and kinases crucial for regulating neuronal firing. Previous studies showed that one such kinase, WEE1-critical to the cell cycle-selectively modulates Nav1.2 channel activity through the accessory protein fibroblast growth factor 14 (FGF14). Here, we tested whether WEE1 exhibits crosstalk with the AKT/GSK3 kinase pathway for coordinated regulation of FGF14/Nav1.2 channel complex assembly and function. Using the in-cell split luciferase complementation assay (LCA), we found that the WEE1 inhibitor II and GSK3 inhibitor XIII reduce the FGF14/Nav1.2 complex formation, while the AKT inhibitor triciribine increases it. However, combining WEE1 inhibitor II with either one of the other two inhibitors abolished its effect on the FGF14/Nav1.2 complex formation. Whole-cell voltage-clamp recordings of sodium currents (INa) in HEK293 cells co-expressing Nav1.2 channels and FGF14-GFP showed that WEE1 inhibitor II significantly suppresses peak INa density, both alone and in the presence of triciribine or GSK3 inhibitor XIII, despite the latter inhibitor's opposite effects on INa. Additionally, WEE1 inhibitor II slowed the tau of fast inactivation and caused depolarizing shifts in the voltage dependence of activation and inactivation. These phenotypes either prevailed or were additive when combined with triciribine but were outcompeted when both WEE1 inhibitor II and GSK3 inhibitor XIII were present. Concerted regulation by WEE1 inhibitor II, triciribine, and GSK3 inhibitor XIII was also observed in long-term inactivation and use dependency of Nav1.2 currents. Overall, these findings suggest a complex role for WEE1 kinase-in concert with the AKT/GSK3 pathway-in regulating the Nav1.2 channelosome.
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Proteínas de Ciclo Celular , Quinase 3 da Glicogênio Sintase , Canal de Sódio Disparado por Voltagem NAV1.2 , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas c-akt , Humanos , Células HEK293 , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Ciclo Celular/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Proteínas Tirosina Quinases/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Multimodality reporter gene imaging combines the sensitivity, resolution and translational potential of two or more signals. The approach has not been widely adopted by the animal imaging community, mainly because its utility in this area is unproven. We developed a new complementation-based reporter gene system where the large component of split NanoLuc luciferase (LgBiT) presented on the surface of cells (TM-LgBiT) interacts with a radiotracer consisting of the high-affinity complementary HiBiT peptide labeled with a radionuclide. Radiotracer uptake could be imaged in mice using SPECT/CT and bioluminescence within two hours of implanting reporter-gene-expressing cells. Imaging data were validated by ex vivo biodistribution studies. Following the demonstration of complementation between the TM-LgBiT protein and HiBiT radiotracer, we validated the use of the technology in the highly specific in vivo multimodal imaging of cells. These findings highlight the potential of this new approach to facilitate the advancement of cell and gene therapies from bench to clinic.
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Genes Reporter , Luciferases , Animais , Camundongos , Luciferases/metabolismo , Luciferases/genética , Humanos , Distribuição Tecidual , Imagem Óptica/métodos , Medições Luminescentes/métodos , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único/métodos , Cintilografia/métodos , Linhagem Celular TumoralRESUMO
BACKGROUND: Metastasis is the main cause of cancer-related deaths, but efficient targeted therapies against metastasis are still missing. Major gaps exist in our understanding of the metastatic cascade, as existing methods cannot combine sensitivity, robustness, and practicality to dissect cancer progression. Addressing this issue requires improved strategies to distinguish early metastatic colonization from metastatic outgrowth. METHODS: Luciferase-labelled MDA-MB-231, MCF7, and 4T1 breast cancer cells were spiked into samples from tumour-naïve mice to establish the limit of detection for disseminated tumour cells. Luciferase-labelled breast cancer cells (±unlabelled cancer-associated fibroblasts; CAFs) were orthotopically implanted in immunocompromised mice. An ex vivo luciferase assay was used to quantify tumour cell dissemination. RESULTS: In vitro luciferase assay confirmed a linear and positive correlation between cancer cell numbers and the bioluminescence detected at single cell level in blood, brain, lung, liver, and mammary fat pad samples. Remarkably, single luciferase-labelled cancer cells were detectable in all of these sites, as the bioluminescence quantified in the analysed samples was substantially higher than background levels. Ex vivo, circulating tumour cells, metastasis, and tumour self-seeding were detected in all samples from animals implanted with highly metastatic luciferase-labelled MDA-MB-231 cells. In turn, detection of poorly metastatic luciferase-labelled MCF7 cells was scarce but significantly enhanced upon co-implantation with CAFs as early as 20 days after the experiment was initiated. CONCLUSIONS: These results demonstrate the feasibility of using an ultrasensitive luciferase-based method to dissect the mechanisms of early metastatic colonization to improving the development of antimetastatic therapies.
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Neoplasias da Mama , Metástase Neoplásica , Células Neoplásicas Circulantes , Animais , Neoplasias da Mama/patologia , Neoplasias da Mama/sangue , Feminino , Camundongos , Humanos , Células Neoplásicas Circulantes/patologia , Células Neoplásicas Circulantes/metabolismo , Modelos Animais de Doenças , Linhagem Celular Tumoral , Detecção Precoce de Câncer/métodos , Luciferases/metabolismoRESUMO
Bioluminescence, the light produced by biochemical reactions involving luciferases in living organisms, has been extensively investigated for various applications. It has attracted particular interest as an internal light source for theranostic applications due to its safe and efficient characteristics that overcome the limited penetration of conventional external light sources. Recent advancements in protein engineering technologies and protein delivery platforms have expanded the application of bioluminescence to a wide range of theranostic areas, including bioimaging, biosensing, photodynamic therapy, and optogenetics. This comprehensive review presents the fundamental concepts of bioluminescence and explores its recent applications across diverse fields. Moreover, it discusses future research directions based on the current status of bioluminescent systems for further expansion of their potential.
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Técnicas Biossensoriais , Medições Luminescentes , Fotoquimioterapia , Nanomedicina Teranóstica , Humanos , Medições Luminescentes/métodos , Nanomedicina Teranóstica/métodos , Animais , Técnicas Biossensoriais/métodos , Fotoquimioterapia/métodos , Optogenética/métodos , Engenharia de Proteínas/métodosRESUMO
The ability of proteins to sense and transmit mechanical forces underlies many biological processes, but characterizing these forces in biological systems remains a challenge. Existing genetically encoded force sensors typically rely on fluorescence or bioluminescence resonance energy transfer (FRET or BRET) to visualize tension. However, these force sensing modules are relatively large, and interpreting measurements requires specialized image analysis and careful control experiments. Here, we report a compact molecular tension sensor that generates a bioluminescent signal in response to tension. This sensor (termed PILATeS) makes use of the split NanoLuc luciferase and consists of the H. sapiens titin I10 domain with the insertion of a 10-15 amino acid tag derived from the C-terminal ß-strand of NanoLuc. Mechanical load across PILATeS mediates exposure of this tag to recruit the complementary split NanoLuc fragment, resulting in force-dependent bioluminescence. We demonstrate the ability of PILATeS to report biologically meaningful forces by visualizing forces at the interface between integrins and extracellular matrix substrates. We further use PILATeS as a genetically encoded sensor of tension experienced by the mechanosensing protein vinculin. We anticipate that PILATeS will provide an accessible means of visualizing molecular-scale forces in biological systems.
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Técnicas Biossensoriais , Luciferases , Medições Luminescentes , Humanos , Luciferases/química , Luciferases/metabolismo , Luciferases/genética , Técnicas Biossensoriais/métodos , Medições Luminescentes/métodos , Conectina/química , Conectina/metabolismo , Vinculina/metabolismo , Vinculina/químicaRESUMO
Bioluminescence imaging (BLI) is an indispensable technique for visualizing the dynamics of diverse biological processes in mammalian animal models, including cancer, viral infections, and immune responses. However, a critical scientific challenge remains: non-invasively visualizing homeostatic and disease mechanisms in freely moving animals to understand the molecular basis of exercises, social behavior, and other phenomena. Classical BLI relies on prolonged camera exposure to accumulate the limited number of photons that traveled from deep tissues in anesthetized or constrained animals. Recent advancements in synthetic bioluminescence reactions, utilizing artificial luciferin-luciferase pairs, have considerably increased the number of detectable photons from deep tissues, facilitating high-speed BLI to capture moving objects. In this review, I provide an overview of emerging synthetic bioluminescence reactions that enable the non-invasive imaging of freely moving animals. This approach holds the potential to uncover unique physiological processes that are inaccessible with current methodologies.
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Medições Luminescentes , Animais , Medições Luminescentes/métodos , Luciferases/metabolismo , Luciferases/genética , HumanosRESUMO
Robinow syndrome is a rare disease caused by variants of seven WNT pathway genes. Craniofacial features include widening of the nasal bridge and jaw hypoplasia. We used the chicken embryo to test whether two missense human FZD2 variants (1301G>T, p.Gly434Val; 425C>T, p.Pro142Lys) were sufficient to change frontonasal mass development. In vivo, the overexpression of retroviruses with wild-type or variant human FZD2 inhibited upper beak ossification. In primary cultures, wild-type and variant human FZD2 significantly inhibited chondrogenesis, with the 425C>T variant significantly decreasing activity of a SOX9 luciferase reporter compared to that for the wild type or 1301G>T. Both variants also increased nuclear shuttling of ß-catenin (CTNNB1) and increased the expression of TWIST1, which are inhibitory to chondrogenesis. In canonical WNT luciferase assays using frontonasal mass cells, the variants had dominant-negative effects on wild-type FZD2. In non-canonical assays, the 425C>T variant failed to activate the reporter above control levels and was unresponsive to exogenous WNT5A. This is the first single amino acid change to selectively alter ligand binding in a FZD receptor. Therefore, FZD2 missense variants are pathogenic and could lead to the altered craniofacial morphogenesis seen in Robinow syndrome.
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Condrogênese , Anormalidades Craniofaciais , Receptores Frizzled , Animais , Embrião de Galinha , Humanos , Bico , beta Catenina/metabolismo , Núcleo Celular/metabolismo , Condrogênese/genética , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/patologia , Nanismo , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Deformidades Congênitas dos Membros , Crânio/patologia , Crânio/embriologia , Proteína 1 Relacionada a Twist/metabolismo , Proteína 1 Relacionada a Twist/genética , Anormalidades Urogenitais , Via de Sinalização WntRESUMO
Crosstalk between metabolism and circadian rhythms is a fundamental building block of multicellular life, and disruption of this reciprocal communication could be relevant to disease. Here, we investigated whether maintenance of circadian rhythms depends on specific metabolic pathways, particularly in the context of cancer. We found that in adult mouse fibroblasts, ATP levels were a major contributor to signal from a clock gene luciferase reporter, although not necessarily to the strength of circadian cycling. In contrast, we identified significant metabolic control of circadian function across a series of pancreatic adenocarcinoma cell lines. Metabolic profiling of congenic tumor cell clones revealed substantial diversity among these lines that we used to identify clones to generate circadian reporter lines. We observed diverse circadian profiles among these lines that varied with their metabolic phenotype: The most hypometabolic line [exhibiting low levels of oxidative phosphorylation (OxPhos) and glycolysis] had the strongest rhythms, while the most hypermetabolic line had the weakest rhythms. Pharmacological enhancement of OxPhos decreased the amplitude of circadian oscillation in a subset of tumor cell lines. Strikingly, inhibition of OxPhos enhanced circadian rhythms only in the tumor cell line in which glycolysis was also low, thereby establishing a hypometabolic state. We further analyzed metabolic and circadian phenotypes across a panel of human patient-derived melanoma cell lines and observed a significant negative association between metabolic activity and circadian cycling strength. Together, these findings suggest that metabolic heterogeneity in cancer directly contributes to circadian function and that high levels of glycolysis or OxPhos independently disrupt circadian rhythms in these cells.
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Ritmo Circadiano , Glicólise , Fosforilação Oxidativa , Neoplasias Pancreáticas , Animais , Humanos , Camundongos , Ritmo Circadiano/fisiologia , Linhagem Celular Tumoral , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/genética , Fibroblastos/metabolismo , Trifosfato de Adenosina/metabolismoRESUMO
Foot-and-mouth disease virus (FMDV) is a highly contagious virus that affects cloven-hoofed animals and causes severe economic losses in the livestock industry. Given that this high-risk pathogen has to be handled in a biosafety level (BSL)-3 facility for safety reasons and the limited availability of BSL-3 laboratories, experiments on FMDV call for more attention. Therefore, we aimed to develop an FMDV experimental model that can be handled in BSL-2 laboratories. The NanoBiT luciferase (Nano-luc) assay is a well-known assay for studying protein-protein interactions. To apply the NanoBiT split luciferase assay to the diagnosis and evaluation of FMD, we developed an inactivated HiBiT-tagged Asia1 Shamir FMDV (AS-HiBiT), a recombinant Asia1 shamir FMDV with HiBiT attached to the VP1 region of Asia1 shamir FMDV. In addition, we established LgBiT-expressing LF-BK cell lines, termed LgBit-LF-BK cells. It was confirmed that inactivated AS-HiBiT infected LgBiT-LF-BK cells and produced a luminescence signal by binding to the intracellular LgBiT of LgBiT-LF-BK cells. In addition, the luminescence signal became stronger as the number of LgBiT-LF-BK cells increased or the concentration of inactivated AS-HiBiT increased. Moreover, we confirmed that inactivated AS-HiBiT can detect seroconversion in sera positive for FMDV-neutralizing antibodies. This NanoBiT split luciferase assay system can be used for the diagnosis and evaluation of FMD and expanded to FMD-like virus models to facilitate the evaluation of FMDV vaccines and antibodies.
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Vírus da Febre Aftosa , Febre Aftosa , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Linhagem Celular , Febre Aftosa/diagnóstico , Febre Aftosa/virologia , Vírus da Febre Aftosa/genética , Luciferases/genética , Luciferases/metabolismoRESUMO
The Takeda G protein-coupled receptor 5 (TGR5) is activated endogenously by primary and secondary bile acids. This receptor is considered a candidate target for addressing inflammatory and metabolic disorders. We have targeted TGR5 with structure-based methods for ligand finding using the recently solved experimental structures, as well as structures obtained from molecular dynamics simulations. Through addressing the orthosteric as well as a putative allosteric site, we identified agonists and positive allosteric modulators. While the predicted binding locations were not in line with their efficacy, our work contributes activating small-molecule ligands that we have thoroughly characterized in vitro.
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Descoberta de Drogas , Receptores Acoplados a Proteínas G , Ligantes , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Humanos , Relação Estrutura-Atividade , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/síntese química , Simulação de Dinâmica Molecular , Relação Dose-Resposta a Droga , Sítio AlostéricoRESUMO
Protein-protein interactions (PPIs) play fundamental roles in many biological processes including the functioning of glycosylation machineries present in the endoplasmic reticulum (ER) and Golgi apparatus of mammalian cells. For the last couple of years, we have been successfully employing the most advanced version of the split luciferase complementation assay, termed NanoBiT, to demonstrate PPIs between solute carrier 35 (SLC35) family members with nucleotide sugar transporting activity and functionally related glycosyltransferases. NanoBiT has several unmatched advantages as compared with other strategies for studying PPIs. Firstly, the tendency of the free luciferase fragments to spontaneously associate is strongly reduced. As a consequence, the fragments of the reconstituted luciferase may dissociate upon the disruption of the PPI of interest. Secondly, the recombinant fusion proteins are expressed at low (near-endogenous) levels. Both of these features significantly minimize the possibility of obtaining false positive results. In this study we pushed the boundaries of this already powerful technique even further by coupling it with bioluminescence imaging of PPIs. Specifically, we visualized homo- and heterologous complexes formed by MGAT1 and MGAT2 glycosylation enzymes tagged with NanoBiT fragments and demonstrated ER-to-Golgi transitions between enzyme homo- and heteromers.
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Gaussia luciferase (GLuc) is one of the most luminescent luciferases known and is widely used as a reporter in biochemistry and cell biology. During catalysis, GLuc undergoes inactivation by irreversible covalent modification. The mechanism by which GLuc generates luminescence and how it becomes inactivated are however not known. Here, we show that GLuc unlike other enzymes has an extensively disordered structure with a minimal hydrophobic core and no apparent binding pocket for the main substrate, coelenterazine. From an alanine scan, we identified two Arg residues required for light production. These residues separated with an average of about 22 Å and a major structural rearrangement is required if they are to interact with the substrate simultaneously. We furthermore show that in addition to coelenterazine, GLuc also can oxidize furimazine, however, in this case without production of light. Both substrates result in the formation of adducts with the enzyme, which eventually leads to enzyme inactivation. Our results demonstrate that a rigid protein structure and substrate-binding site are no prerequisites for high enzymatic activity and specificity. In addition to the increased understanding of enzymes in general, the findings will facilitate future improvement of GLuc as a reporter luciferase.
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Luciferases , Luciferases/química , Luciferases/metabolismo , Luciferases/genética , Animais , Luminescência , Copépodes/enzimologia , Modelos Moleculares , Imidazóis/química , Imidazóis/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Pirazinas/química , Pirazinas/metabolismoRESUMO
Bioluminescence imaging has become an essential non-invasive tool in cancer research for monitoring various cellular processes and tumor progression in vivo. In this article, we aimed to propose a transduction and selection protocol for reliable in vivo bioluminescent measurements in immunocompetent mouse models. Using two different heterogenous luciferase-expressing cell models, we underlined factors influencing transduction. The protocol was tested through an in vitro luciferase activity assay as well as using in vivo longitudinal monitoring of metastases formation (In Vivo Imaging System®). The data were cross validated with histological assessment. Our results demonstrated stable and proportional in vitro and in vivo bioluminescent signals correlating with actual metastatic burden. Furthermore, ex vivo analysis confirmed the accuracy of bioluminescent imaging in quantifying metastatic surface area. This protocol should ensure reliable and reproducible measurements in cancer research utilizing luciferase-positive cell lines, confirming the validity and accuracy of preclinical studies in immunocompetent models.
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Rift Valley fever virus is able to infect multiple organs and cell types, and the course of infection varies between viral strains and between individuals in particular according to age, genetic background, and physiological status. Studies on viral and host factors involve detecting and quantifying viral load at multiple time points and in multiple tissues. While this is classically performed by genome quantification or viral titration, in vivo imaging techniques using recombinant viruses expressing a bioluminescent or fluorescent protein allow noninvasive longitudinal studies on the same group of mice over the entire course of disease and the detection of unsuspected sites of infection. Here, we describe the protocol to monitor and characterize mouse infection with Rift Valley fever virus by in vivo imaging using recombinant viruses expressing light-emitting reporter genes.
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Genes Reporter , Medições Luminescentes , Vírus da Febre do Vale do Rift , Animais , Camundongos , Medições Luminescentes/métodos , Vírus da Febre do Vale do Rift/genética , Febre do Vale de Rift/virologia , Febre do Vale de Rift/diagnóstico , Carga Viral/métodos , Modelos Animais de Doenças , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismoRESUMO
Recent advances in molecular modeling using deep learning can revolutionize our understanding of dynamic protein structures. NMR is particularly well-suited for determining dynamic features of biomolecular structures. The conventional process for determining biomolecular structures from experimental NMR data involves its representation as conformation-dependent restraints, followed by generation of structural models guided by these spatial restraints. Here we describe an alternative approach: generating a distribution of realistic protein conformational models using artificial intelligence-(AI-) based methods and then selecting the sets of conformers that best explain the experimental data. We applied this conformational selection approach to redetermine the solution NMR structure of the enzyme Gaussia luciferase. First, we generated a diverse set of conformer models using AlphaFold2 (AF2) with an enhanced sampling protocol. The models that best-fit NOESY and chemical shift data were then selected with a Bayesian scoring metric. The resulting models include features of both the published NMR structure and the standard AF2 model generated without enhanced sampling. This "AlphaFold-NMR" protocol also generated an alternative "open" conformational state that fits nearly as well to the overall NMR data but accounts for some NOESY data that is not consistent with first "closed" conformational state; while other NOESY data consistent with this second state are not consistent with the first conformational state. The structure of this "open" structural state differs from that of the "closed" state primarily by the position of a thumb-shaped loop between α-helices H5 and H6, revealing a cryptic surface pocket. These alternative conformational states of Gluc are supported by "double recall" analysis of NOESY data and AF2 models. Additional structural states are also indicated by backbone chemical shift data indicating partially-disordered conformations for the C-terminal segment. Considered as a multistate ensemble, these multiple states of Gluc together fit the NOESY and chemical shift data better than the "restraint-based" NMR structure and provide novel insights into its structure-dynamic-function relationships. This study demonstrates the potential of AI-based modeling with enhanced sampling to generate conformational ensembles followed by conformer selection with experimental data as an alternative to conventional restraint satisfaction protocols for protein NMR structure determination.
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Bacillus subtilis is a Gram-positive bacterium that is frequently used in the bioindustry for the production of various proteins, because of its superior protein secretion capacities. To determine optimal conditions for protein secretion by B. subtilis, a quick and sensitive method for measuring protein secretion is crucial. A fast and universal assay is most useful for detecting diverse proteins in a high-throughput manner. In this study, we introduce a split-luciferase-based method for measuring protein secretion by B. subtilis. The NanoBiT system was used to monitor secretion of four different proteins: xylanase A, amylase M, protein glutaminase A, and GFP nanobody. Our findings underscore the split-luciferase system as a quick, sensitive, and user-friendly method.
Assuntos
Bacillus subtilis , Proteínas de Bactérias , Bacillus subtilis/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Luciferases/metabolismo , Luciferases/genética , Endo-1,4-beta-Xilanases/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Transporte Proteico , Amilases/metabolismo , Glutaminase/metabolismoRESUMO
Renilla luciferase catalyzes the oxidation of coelenterazine to coelenteramide and results in the emission of a photon of light. Although Renilla luciferase has various applications in biotechnology, its low thermal stability limits the development of its applications. Arginine is a well-known stabilizing amino acid that plays a key role in protein stabilization against inactivation. However, its impact on enzyme properties is unpredictable. This study investigates the impact of arginine on the kinetics and thermal stability of Renilla luciferase. The enzyme's performance was significantly enhanced in the presence of arginine, with catalytic efficiency increasing by 3.31-fold and 3.08-fold when exposed to 0.2 M and 0.3 M arginine, respectively. Additionally, arginine improved the thermal stability of Renilla luciferase. Molecular dynamics simulation showed that the addition of 0.2 M arginine reduced the binding of coelenteramide, the reaction product and an enzyme inhibitor, to the active site of the Renilla luciferase. Therefore, the release of the product was accelerated, and the affinity of Renilla luciferase for coelenterazine increased. Furthermore, Molecular dynamics studies indicated an increased network of water molecules surrounding Renilla luciferase in the presence of 0.2 M arginine. This network potentially enhances the hydrophobic effect on the protein structure, ultimately improving enzyme stability. The findings of this study hold promise for the development of commercial kits incorporating Renilla luciferase.
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Agrobacterium-mediated transient expression methods are widely used to study gene function in both model and non-model plants. Using a dual-luciferase assay, we quantified the effect of Agrobacterium-infiltration parameters on the transient transformation efficiency of Catharanthus roseus seedlings. We showed that transformation efficiency is highly sensitive to seedling developmental state and a pre- and post-infiltration dark incubation and is less sensitive to the Agrobacterium growth stage. For example, 5 versus 6 days of germination in the dark increased seedling transformation efficiency by seven- to eight-fold while a dark incubation pre- and post-infiltration increased transformation efficiency by five- to 13-fold. Agrobacterium in exponential compared with stationary phase increased transformation efficiency by two-fold. Finally, we quantified the variation in our Agrobacterium-infiltration method in replicate infiltrations and experiments. Within a given experiment, significant differences of up to 2.6-fold in raw firefly luciferase (FLUC) and raw Renilla luciferase (RLUC) luminescence occurred in replicate infiltrations. These differences were significantly reduced when FLUC was normalized to RLUC values, highlighting the utility of including a reference reporter to minimize false positives. Including a second experimental replicate further reduced the potential for false positives. This optimization and quantitative validation of Agrobacterium infiltration in C. roseus seedlings will facilitate the study of this important medicinal plant and will expand the application of Agrobacterium-mediated transformation methods in other plant species.
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Glioblastoma (GBM) is the most aggressive brain cancer. To model GBM in research, orthotopic brain tumor models, including syngeneic models like GL261 and genetically engineered mouse models like TRP, are used. In longitudinal studies, tumor growth and the treatment response are typically tracked with in vivo imaging, including bioluminescence imaging (BLI), which is quick, cost-effective, and easily quantifiable. However, BLI requires luciferase-tagged cells, and recent studies indicate that the luciferase gene can elicit an immune response, leading to tumor rejection and experimental variation. We sought to optimize the engraftment of two luciferase-expressing GBM models, GL261 Red-FLuc and TRP-mCherry-FLuc, showing differences in tumor take, with GL261 Red-FLuc cells requiring immunocompromised mice for 100% engraftment. Immunohistochemistry and MRI revealed distinct tumor characteristics: GL261 Red-FLuc tumors were well-demarcated with densely packed cells, high mitotic activity, and vascularization. In contrast, TRP-mCherry-FLuc tumors were large, invasive, and necrotic, with perivascular invasion. Quantifying the tumor volume using the HALO® AI analysis platform yielded results comparable to manual measurements, providing a standardized and efficient approach for the reliable, high-throughput analysis of luciferase-expressing tumors. Our study highlights the importance of considering tumor engraftment when using luciferase-expressing GBM models, providing insights for preclinical research design.
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BACKGROUND/AIM: Genetic reporters encoding fluorescent proteins or luciferase have been used in vivo for the last three decades with claims about their superiority or inferiority over each other. In the present report, a head-to-head in vivo comparison of green fluorescent protein (GFP) fluorescence imaging and luciferase-luciferin imaging, using single-nanometer laser-excitation tuning of fluorescence excitation and an ultra-low-light-detection camera and optics was performed. MATERIALS AND METHODS: Mouse Lewis-lung carcinoma cells labeled with GFP (LLC-GFP) or luciferase (LL/2-Luc2) were injected subcutaneously into the flank of nude mice. One week after injection, GFP-fluorescence imaging and luciferase-luciferin imaging was performed using the UVP Biospectrum Advanced system with excitation at 487 nm and peak emission at 513 nm for GFP, and with emission at 560 nm for luciferase-luciferin. GFP fluorescence images were obtained at 0, 10, and 20 min. Luciferase-luciferin images were obtained 10 and 20 min after the injection of D-luciferin. RESULTS: The intensity of GFP images was 55,909 at 0 min, 56,186 at 10 min, and 57,085 at 20 min, and maintained after 20 min. The intensity of luciferase-luciferin images was 28,065 at 10 min after the injection of D-luciferin and 5,199 at 20 min after the injection. The intensity of luciferase-luciferin images decreased by approximately 80% at 20 min compared to 10 min. An exposure time of 30 s for luciferase-luciferin imaging was needed compared to 100 ms for GFP fluorescence imaging in order to detect signals. CONCLUSION: An imaging system with single-nanometer tuning fluorescence excitation and an ultra-low-light detection camera and optics was able to directly visualize both GFP and luciferase-luciferin images in vivo. The intensity and stability of the signals were both greater for GFP than for luciferase-luciferin, and the exposure time for GFP was 300 times faster, demonstrating the superiority of GFP.