In Embryo Gene Reporter Assays for Evaluation of Cis-Regulatory Regions.

Gene expression reporter assays measure the relevance of cis-regulatory elements and DNA-binding proteins in modulating transcriptional activity. Commonly, they are performed in cell lines. However, regulation of transcriptional activity during development is complex and dynamic, and not many cell lines reproduce the embryonic conditions. Thus, conclusions derived from cell line data provide limited information about embryonic development. On the other hand, one of the major hurdles for embryonic assays is delivering reporter plasmids in a tissue-specific manner. In this sense, the chick embryo is a good model system to perform these assays. Electroporation of chick embryos provides temporal and spatially controlled plasmid delivery. Further, it is a well-established, easy, and an economical procedure. Here, we describe in detail how to measure in the chick neural tube (1) enhancer activity with GFP, (2) enhancer activity with luciferase, and (3) 3'UTR activity with luciferase.

Evaluating Single-Nucleotide Variants in MicroRNA Targeting Sites and Mature MicroRNA In Vitro Cell Culture by Luciferase Reporter Gene Assays.

MicroRNAs (miRs) are small non-coding RNAs of 21-24 nucleotides in length that modulate gene expression by targeting the untranslated region (UTR) of mRNA. Single-nucleotide variants (SNVs) in primary miRs (pri-miRs), precursor miRs (pre-miRs), promoters of pri-miRs, and seed regions can affect miR stability or processing, may influence mature miR expression, and can affect target gene identification, respectively. The present protocol tests the binding and activity of miRs on 3'-UTR target sequences based on the expression of luciferase as a reporter gene fused to the UTR sequence in the presence of plasmids containing pre-miR of interest to test in vitro cell culture assay.

A Simple Bioluminescent Assay for the Screening of Cytotoxic Molecules Against the Intracellular Form of Leishmania infantum.

This chapter describes a viability assay for the intracellular (amastigote) and clinically relevant form of Leishmania infantum that is based on the detection of bioluminescence (BL) signal. The assay uses a reporter cell line of L. infantum that expresses constitutively a redshifted luciferase from Photinus pyralis and murine macrophages (cell line J774.A1) as host cells for infection. The host cell line was selected because it is a differentiated cell line, easy to manipulate in vitro, and advantageous for ethical reasons. This chapter introduces an assay designed for the screening of bioactive compounds/molecules employing a 96-well microplate and a 24 h treatment. The assay setup shows excellent balance between simplicity (cell culture manipulation/infection and timing) and quality parameters, as well as potential to detect drug-like molecules acting in a fast and cytotoxic manner.

A Simple, Robust, and Affordable Bioluminescent Assay for Drug Screening Against Infective African Trypanosomes.

This chapter introduces a simple and robust in vitro viability assay to screen bioactive small molecules (e.g., natural, synthetic) against the monomorphic and infective (bloodstream) form of Trypanosoma brucei brucei. The assay relies on a bioluminescent transgenic parasite harboring a genetically encoded copy of a thermostable redshifted firefly luciferase from Photinus pyralis.The major advantages of the assay are simplicity and cost efficiency, along with excellent quality parameters. The bioassay allows estimating parasite numbers and viability (and metabolic state) as a function of bioluminescence (BL) signal. Parasites are grown in the presence of the molecules of interest in a 96-well microplate, and 24 h later, BL is determined with a simple protocol lacking washing steps, using cost-efficient reagents with a reasonable readout time for high-throughput applications.

Cloning and molecular properties of a novel luciferase from the Brazilian Bicellonycha lividipennis (Lampyridae: Photurinae) firefly: comparison with other firefly luciferases.

Several firefly luciferases eliciting light emission in the yellow-green range of the spectrum and with distinct kinetic properties have been already cloned, sequenced, and characterized. Some of them are currently being applied as analytical reagents and reporter genes for bioimaging and biosensors, and more recently as potential color tuning indicators of intracellular pH and toxic metals. They were cloned from the subfamilies Lampyrinae (Photinini: Photinus pyralis, Macrolampis sp2; Cratomorphini: Cratomorphus distinctus), Photurinae (Photuris pennsylvanica), Luciolinae (Luciola cruciata, L. lateralis, L. mingrelica, L. italica, Hotaria parvula), and Amydetinae (Amydetes vivianii) occurring in different parts of the world. The largest number has been cloned from fireflies occurring in Brazilian biomes. Taking advantage of the large biodiversity of fireflies occurring in the Brazilian Atlantic rainforest, here we report the cloning and characterization of a novel luciferase cDNA from the Photurinae subfamily, Bicellonycha lividipennis, which is a very common firefly in marshlands in Brazil. As expected, multialignements and phylogenetic analysis show that this luciferase clusters with Photuris pennsylvanica adult isozyme, and with other adult lantern firefly luciferases, in reasonable agreement with traditional phylogenetic analysis. The luciferase elicits light emission in the yellow-green region, has kinetics properties similar to other adult lantern firefly luciferases, including pH- and metal sensitivities, but displays a lower sensitivity to nickel, which is suggested to be caused by the natural substitution of H310Y.

Real-time monitoring of Metridia luciferase release from cells upon interaction with model toxic substances by a fully automatic flow setup - A proof of concept.

This manuscript reports on a fully automatic sequential injection system incorporating a 3D printed module for real-time monitoring of the release of Metridia luciferase from a modified liver epithelial cell line. To this end, a simple and effective approach for the automation of flash-type chemiluminescence assays was developed. The 3D printed module comprised an apical and a basal compartment that enabled monitoring membrane processes on both sides of the cell monolayer aimed at elucidating the direction of luciferase release. A natural release was observed after transfection with the luciferase plasmid by online measurement of the elicited light from the reaction of the synthesized luciferase with the coelenterazine substrate. Model substances for acute toxicity from the group of cholic acids - chenodeoxycholic and deoxycholic acids - were applied at the 1.0 and 0.5 mmol L-1 levels. The tested cholic acids caused changes in cell membrane permeability that was accompanied by an increased luciferase release. The obtained kinetic profiles were evaluated based on the delay between the addition of the toxic substance and the increase of the chemiluminescence signal. All experiments were carried out in a fully automatic system in ca. 5 min per sample in 30 min intervals and no manual interventions were needed for a sampling period of at least 6 h.

A simple, robust, and affordable bioluminescent assay for drug discovery against infective African trypanosomes.

African trypanosomiasis is a major problem for human and animal health in endemic countries, where it threatens millions of people and affects economic development. New drugs are needed to overcome the toxicity, administration, low efficacy, and resistance issues of the current chemotherapy. Robust, simple, and economical high-throughput, whole-cell-based assays are required to accelerate the identification of novel chemical entities. With this aim, we generated a bioluminescent cell line of the bloodstream stage of Trypanosoma brucei brucei and established a screening assay. Trypanosomes were stably transfected to constitutively express a thermostable red-shifted luciferase. The growth phenotype and drug sensitivity of the reporter cell line were essentially identical to that of the parental cell line. The endogenous luciferase activity, measured by a simple bioluminescence assay, proved to be proportional to parasite number and metabolic status. The assay, optimized to detect highly potent compounds in a 96-well-plate format, was validated by screening a small compound library (inter-assay values for Z' factor and coefficient variation were 0.77 and 5.8%, respectively). With a hit-confirmation ratio of ~97%, the assay was potent enough to identify several hits with EC50 ≤ 10 µM. Preliminary tests indicated that the assay can be scaled up to a 384-well-plate format without compromising its robustness. In summary, we have generated reporter trypanosomes and a simple, robust, and affordable bioluminescence screening assay with great potential to speed up the early-phase drug discovery against African trypanosomes.

A Novel Brighter Bioluminescent Fusion Protein Based on ZZ Domain and Amydetes vivianii Firefly Luciferase for Immunoassays.

Immunoassays are widely used for detection of antibodies against specific antigens in diagnosis, as well as in electrophoretic techniques such as Western Blotting. They usually rely on colorimetric, fluorescent or chemiluminescent methods for detection. Whereas the chemiluminescence methods are more sensitive and widely used, they usually suffer of fast luminescence decay. Here we constructed a novel bioluminescent fusion protein based on the N-terminal ZZ portion of protein A and the brighter green-blue emitting Amydetes vivianii firefly luciferase. In the presence of D-luciferin/ATP assay solution, the new fusion protein, displays higher bioluminescence activity, is very thermostable and produces a sustained emission (t1/2 > 30 min). In dot blots, we could successfully detect rabbit IgG against firefly luciferases, Limpet Haemocyanin, and SARS-CoV-2 Nucleoprotein (1-250 ng), as well as the antigen bound antibodies using either CCD imaging, and even photography using smartphones. Using CCD imaging, we could detect up to 100 pg of SARS-CoV-2 Nucleoprotein. Using this system, we could also successfully detect firefly luciferase and SARS-CoV-2 nucleoprotein in Western Blots (5-250 ng). Comparatively, the new fusion protein displays slightly higher and more sustained luminescent signal when compared to commercial HRP-labeled secondary antibodies, constituting a novel promising alternative for Western Blotting and immunoassays.

A global search for novel transcription factors impacting the Neurospora crassa circadian clock.

Eukaryotic circadian oscillators share a common circuit architecture, a negative feedback loop in which a positive element activates the transcription of a negative one that then represses the action of the former, inhibiting its own expression. While studies in mammals and insects have revealed additional transcriptional inputs modulating the expression of core clock components, this has been less characterized in the model Neurospora crassa, where the participation of other transcriptional components impacting circadian clock dynamics remains rather unexplored. Thus, we sought to identify additional transcriptional regulators modulating the N. crassa clock, following a reverse genetic screen based on luminescent circadian reporters and a collection of transcription factors (TFs) knockouts, successfully covering close to 60% of them. Besides the canonical core clock components WC-1 and -2, none of the tested transcriptional regulators proved to be essential for rhythmicity. Nevertheless, we identified a set of 23 TFs that when absent lead to discrete, but significant, changes in circadian period. While the current level of analysis does not provide mechanistic information about how these new players modulate circadian parameters, the results of this screen reveal that an important number of light and clock-regulated TFs, involved in a plethora of processes, are capable of modulating the clockworks. This partial reverse genetic clock screen also exemplifies how the N. crassa knockout collection continues to serve as an expedite platform to address broad biological questions.

Selective inhibition of Zophobas morio (Coleoptera: Tenebrionidae) luciferase-like enzyme luminescence by diclofenac and potential suitability for light-off biosensing.

The accumulation of toxic carboxylic compounds may cause severe effects on the environment and living organisms. A luciferase-like enzyme, previously cloned from the Malpighian tubules of the non-luminescent Zophobas morio mealworm, displays thioesterification activity with a wide range of carboxylic substrates, and produces weak red luminescence in the presence of ATP and firefly d-luciferin, a xenobiotic for this organism. To better investigate the function of this enzyme in carboxylic xenobiotic detoxification, we analyzed the inhibitory effect of different xenobiotic carboxylic acids on the luminescence activity of this enzyme, including environmental pollutants and pharmaceutical compounds. Noteworthy, the anti-inflammatory drug diclofenac severely inhibited this luciferase-like enzyme luminescence activity, both in in vitro (IC50 20 µM) and in vivo in bacterial cells assays, when compared with other beetle luciferases. Similar results were obtained with its brighter I327S mutant. Kinetic analysis of diclofenac's effect on luminescence activity indicated mixed-type inhibition for both ATP and d-luciferin. Modelling studies showed five potential binding sites for diclofenac, including the coenzyme A binding site, which showed one of the highest binding constant. Taken together, these results raise the possibility of using this luciferase-like enzyme for the development of novel whole-cell luminescent biosensors for diclofenac and similar drugs.

A novel brighter bioluminescent fusion protein based on ZZ domain and amydetes vivianii firefly luciferase for immunoassays

Immunoassays are widely used for detection of antibodies against specific antigens in diagnosis, as well as in electrophoretic techniques such as Western Blotting. They usually rely on colorimetric, fluorescent or chemiluminescent methods for detection. Whereas the chemiluminescence methods are more sensitive and widely used, they usually suffer of fast luminescence decay. Here we constructed a novel bioluminescent fusion protein based on the N-terminal ZZ portion of protein A and the brighter green-blue emitting Amydetes vivianii firefly luciferase. In the presence of D-luciferin/ATP assay solution, the new fusion protein, displays higher bioluminescence activity, is very thermostable and produces a sustained emission (t1/2 > 30 min). In dot blots, we could successfully detect rabbit IgG against firefly luciferases, Limpet Haemocyanin, and SARS-CoV-2 Nucleoprotein (1–250 ng), as well as the antigen bound antibodies using either CCD imaging, and even photography using smartphones. Using CCD imaging, we could detect up to 100 pg of SARS-CoV-2 Nucleoprotein. Using this system, we could also successfully detect firefly luciferase and SARS-CoV-2 nucleoprotein in Western Blots (5–250 ng). Comparatively, the new fusion protein displays slightly higher and more sustained luminescent signal when compared to commercial HRP-labeled secondary antibodies, constituting a novel promising alternative for Western Blotting and immunoassays.

Functional evaluation of human papillomavirus type 31 long control region variants.

Persistent infections by high-risk human papillomavirus (HR-HPV) are a necessary condition, but not sufficient for cervical cancer development. Genetic variants of HR-HPV appear to be related to the risk of persistent infections. The study performed a functional evaluation of variants of the HPV-31 promoter region (LCR). For this, cloning and subcloning of variants HPV-31/UFPE-21 HPV-31/UFPE-89, HPV-31/UFPE-66, E2 gene and prototype HPV-31 were performed. Transfection with different concentrations of E2 was done and the concentration of 25 ng was determined to be ideal for LCR activation. HPV-31/UFPE-21 and HPV-31/UFPE-89 have a greater ability to alter Nluc reporter gene expression levels and HPV-31/UFPE-66 showed decreased levels of gene expression of Nluc reporter gene compared to control. Statistical analysis showed a significant difference between the polymorphic LCR regions and the control (p < 0.0001). A more refined profile of variants of HPV-31 and its importance for the prognosis of cervical lesions begins to be drawn.

Evaluation of Phenolic Compound Toxicity Using a Bioluminescent Assay with the Fungus Gerronema viridilucens.

Basidiomycetes (phylum Basidiomycota) are filamentous fungi characterized by the exogenous formation of spores on a club-shaped cell called a basidium that are often formed on complex fruiting bodies (mushrooms). Many basidiomycetes serve an important role in recycling lignocellulosic material to higher trophic levels, and some show symbiotic relationships with plants. All known bioluminescent fungi are mushroom-forming basidiomycetes in the order Agaricales. Hence, the disruption of the basidiomycete community can entirely compromise the carbon cycle in nature from fungi to higher trophic levels. The fungus Gerronema viridilucens was used in the present study to investigate the toxicity of a phenolic compound series based on the inhibition of its bioluminescence. The median effect concentration (EC50) obtained from curves of bioluminescence inhibition versus log [phenolic compound] showed that 2,4,6-trichlorophenol was the most toxic compound in the series. The log EC50 values of all phenolic compounds were then used for the prediction of their toxicity. The univariate correlation of log EC50 values obtained from 6 different phenolic compounds was stronger with the dissociation constant (pKa ) than with 1-octanol/water partition coefficient (KOW ). Nevertheless, the toxicity can be better predicted by using both parameters, suggesting that the phenol-driven uncoupling of fungus mitochondrial adenosine triphosphate synthesis is the origin of phenolic compound toxicity to the test fungus. Environ Toxicol Chem 2020;39:1558-1565. © 2020 SETAC.

A circadian clock in Neurospora crassa functions during plant cell wall deconstruction.

Circadian clocks are autonomous timers that are believed to confer organisms a selective advantage by enabling processes to occur at appropriate times of the day. In the model fungus Neurospora crassa, 20-40 % of its genes are reported to be under circadian regulation, as assayed in simple sugar media. Although it has been well-described that Neurospora efficiently deconstructs plant cell wall components, little is known regarding the status of the clock when Neurospora grows on cellulosic material, or whether such a clock has an impact on any of the genes involved in this process. Through luciferase-based reporters and fluorescent detection assays, we show that a clock is functioning when Neurospora grows on cellulose-containing wheat straw as the only carbon and nitrogen source. Additionally, we found that the major cellobiohydrolase encoding gene involved in plant cell wall deconstruction, cbh-1, is rhythmically regulated by the Neurospora clock, in a manner that depends on cellulose concentration and on the transcription factor CRE-1, known as a key player in carbon-catabolite repression in this fungus. Our findings are a step towards a more comprehensive understanding on how clock regulation modulates cellulose degradation, and thus Neurospora's physiology.

Evaluation of NanoLuc, RedLuc and Luc2 as bioluminescent reporters in a cutaneous leishmaniasis model.

New drugs for the treatment of human leishmaniasis are urgently needed, considering the limitations of current available options. However, pre-clinical evaluation of drug candidates for leishmaniasis is challenging. The use of luciferase-expressing parasites for parasite load detection is a potentially powerful tool to accelerate the drug discovery process. We have previously described the use of Leishmania amazonensis mutants expressing firefly luciferase (Luc2) for drug testing. Here, we describe three new mutant L. amazonensis lines that express different variants of luciferases: NanoLuc, NanoLuc-PEST and RedLuc. These mutants were evaluated in drug screening protocols. NanoLuc-parasites, in spite of high bioluminescence intensity in vitro, were shown to be inadequate in discriminating between live and dead parasites. Bioluminescence detection from intracellular amastigotes expressing NanoLuc-PEST, RedLuc or Luc2 proved more reliable than microscopy to determine parasite killing. Increased sensitivity was observed in vivo with RedLuc-expressing parasites as compared to NanoLuc-expressing L. amazonensis. Our data indicates that NanoLuc is not suitable for in vivo parasite burden determination. Additionally, RedLuc and the conventional luciferase Luc2 demonstrated equivalent sensitivity in an in vivo model of cutaneous leishmaniasis.

The luciferase reporter system of the MMP12 endogenous promoter for investigating transcriptional regulation of the human MMP12 gene

Background: Matrix metalloproteinase 12 (MMP12), a member of MMPs, can take lots of roles including extracellular matrix component degradation, viral infection, inflammation, tissue remodeling and tumorigenesis. To explore the transcriptional regulation of MMP12 gene, a sensitive luciferase reporter HEK293 cell line for endogenous MMP12 promoter was generated by CRISPR/Cas9 technology. Results: The HEK293-MMP12-T2A-luciferase-KI cell line was successfully established by CRISPR/Cas9 technology. The sequencing results indicated that one allele of the genome was proven to have a site-directed insertion of luciferase gene and another allele of the genome was confirmed to have additional 48 bp insertion in this cell line. The cell line was further demonstrated to be a sensitive reporter of the endogenous MMP12 promoter by applying transcription factors STAT3, AP-1 and SP-1 to the cell line. The reporter cell line was then screened with bioactive small molecule library, and a small molecule Tanshinone I was found to significantly inhibit the transcriptional activity of MMP12 gene in HEK293-MMP12-T2A-luciferase-KI cell line by luciferase activity assay, which was further confirmed to inhibit the expression of MMP12 mRNA in wild-type HEK293 cells. Conclusions: This novel luciferase knock-in reporter system will be helpful for investigating the transcriptional regulation of MMP12 gene and screening the drugs targeting MMP12 gene.

Generation of Tumor Cells Expressing Firefly Luciferase (fLuc) to Evaluate the Effectiveness of CAR in a Murine Model.

Immunotherapy has been showed as a promisor treatment, in special for hematological diseases. Chimeric antigen receptor T cells (CARs) which are showing satisfactory results in early-phase cancer clinical trials can be highlighted. However, preclinical models are critical steps prior to clinical trial. In this way, a well-established preclinical model is an important key in order to confirm the proof of principle. For this purpose, in this chapter will be pointed the methods to generate tumor cells expressing firefly Luciferase. In turn, these modified cells will be used to create a subcutaneous and a systemic murine model of Burkitt's lymphoma in order to evaluate the effectiveness of CAR-T.

Generation and Characterization of a Dual-Reporter Transgenic Leishmania braziliensis Line Expressing eGFP and Luciferase.

In this study, we generated a transgenic strain of Leishmania braziliensis, an etiological agent associated with a diversity of clinical manifestations of leishmaniasis ranging from localized cutaneous to mucocutaneous to disseminated disease. Transgenic parasites expressing reporter proteins are valuable tools for studies of parasite biology, host-pathogen interactions, and anti-parasitic drug development. To this end, we constructed an L. braziliensis line stably expressing the reporters eGFP and luciferase (eGFP-LUC L. braziliensis). The integration cassette co-expressing the two reporters was targeted to the ribosomal locus (SSU) of the parasite genome. Transgenic parasites were characterized for their infectivity and stability both in vitro and in vivo. Parasite maintenance in axenic long-term culture in the absence of selective drugs did not alter expression of the two reporters or infection of BALB/c mice, indicating stability of the integrated cassette. Infectivity of eGFP-LUC, L. braziliensis, both in vivo and in vitro was similar to that obtained with the parental wild type strain. The possibility of L. braziliensis tracking and quantification using fluorescence and luminescence broadens the scope of research involving this neglected species, despite its importance in terms of public health concerning the leishmaniasis burden.

Ex vivo evaluation of intravitreal mesenchymal stromal cell viability using bioluminescence imaging.

BACKGROUND: Bone marrow-derived mesenchymal stromal cell (MSC) therapy is a promising treatment for several degenerative ocular diseases; however, no reproducible method of monitoring these cells into the eye has been established. The aim of this study was to describe successful bioluminescence imaging (BLI) to detect viable luciferase-expressing MSC in the eye. METHODS: Human donor MSC in culture were transduced with 50 µl luciferase lentiviral vector (three viral particles/cell) prior to intraocular injection. Twenty-one right eyes of 21 rabbits were evaluated through BLI after receiving 1 × 106 luciferase-expressing MSC intravitreally. Contralateral eyes were injected with vehicle (phosphate-buffered saline (PBS)) and were used as controls. At seven different time points (1 h to 60 days), D-luciferin (40 mg/ml, 300 µl PBS) was injected in subsets of six enucleated eyes for evaluation of radiance decay through BLI analysis. CD90 and CD73 immunofluorescence was studied in selected eyes. RESULTS: Eyes injected with MSC showed high BLI radiance immediately after D-luciferin injection and progressive decay until 60 days. Mean BLI radiance measures from eyes with luciferase-expressing MSC were significantly higher than controls from 8 h to 30 days. At the thirtieth day, positive CD90- and CD73-expressing cells were observed only in the vitreous cavity of eyes injected with MSC. CONCLUSIONS: Viable MSC were identified in the vitreous cavity 1 month after a single injection. Our results confirmed BLI as a useful and reliable method to detect MSC injected into the eye globe.

Comparison of the thermostability of recombinant luciferases from Brazilian bioluminescent beetles: Relationship with kinetics and bioluminescence colours.

Firefly luciferases have been used extensively as bioanalytical reagents and their cDNAs as reporter genes for biosensors and bioimaging, but they are in general unstable at temperatures above 30°C. In the past few years, efforts have been made to stabilize some firefly luciferases for better application as analytical reagents. Novel luciferases from different beetle families, displaying distinct bioluminescence colours and kinetics, may offer desirable alternatives to extend the range of applications. In the past years, our group has cloned the largest variety of luciferases from the three main families of bioluminescent beetles (Elateridae: P. termitilluminans, F. bruchi, P. angustus; Phengodidae: P. hirtus, P. vivianii; and Lampyridae: A. vivianii, C. distinctus and Macrolampis sp2) occurring in Brazilian biomes. We compared the thermostability of these recombinant luciferases and investigated their relationships with bioluminescence spectra and kinetics. The most thermostable luciferases were those of Pyrearinus termitilluminans larval click beetle (534 nm), Amydetes vivianii firefly (539 nm) and Phrixotrix vivianii railroad worm (546 nm), which are the most blue-shifted examples in each family, confirming the trend that the most blue-shifted emitting luciferases are also the most thermostable. Comparatively, commercial P. pyralis firefly luciferase was less thermostable than P. termitilluminans click beetle and A. vivianii firefly luciferases. The higher thermostability in these luciferases could be related to higher degree of hydrophobic packing and disulfide bond content (for firefly luciferases).