cDNA cloning and characterization of a secreted luciferase from the luminous Japanese ostracod, Cypridina noctiluca

A secreted luciferase from the marine ostracod, Vargula hilgendorfii, is a useful tool for gene expression assays in living mammalian cells. We have cloned the cDNA of a new secreted luciferase from the ostracod Cypridina noctiluca, which inhabits the coast of Japan. C. noctiluca luciferase consists of 553 amino acid residues with a molecular mass of 61,415 Da, as deduced from the nucleotide sequence. The homologies of nucleotide and amino acid sequences with V. hilgendorfii luciferase are 79.2% and 83.1%, respectively. C. noctiluca luciferase can expressed in and secreted from cultured mammalian cells. The characteristic properties of expressed C. noctiluca luciferase are similar to those of V. hilgendorfii luciferase. However, the activity of C. noctiluca luciferase in culture medium is much higher than that of V. hilgendorfii luciferase, suggesting that C. noctiluca luciferase is a highly potent reporter enzyme for real-time and continuous monitoring of gene expression in living cells.     

Modulation of firefly luciferase stability and impact on studies of gene regulation

Two of the reporter enzymes most commonly used in studies of eukaryotic gene expression are chloramphenicol acetyl-transferase (CAT) and firefly luciferase (Luc). CAT has a half-life of about 50 h in mammalian cells, making it useful for transient transfection assays but less suitable for assays with stable cell lines. Luc has a half-life of only 3 h in mammalian cells, making it much more responsive in stable cell lines. Luc instability arises from its sensitivity to proteolysis both in vivo and in vitro. Compounds that resemble its natural substrate, luciferin, act as effective competitive inhibitors in vitro. When these compounds (e.g., phenylbenzothiazole) are added to either prokaryotic or eukaryotic cells, more than tenfold increases in Luc activity can be observed. This increased activity results from a lower rate of degradation of the enzyme in vivo and can be mimicked in vitro as phenylbenzothiazole protects Luc from trypsin digestion while it has no effect on the rate of digestion of alkaline phosphatase.     

Firefly luciferase luminescence assays using scintillation counters for quantitation in transfected mammalian cells

The firefly enzyme luciferase catalyzes the luminescent reaction of luciferin with ATP and oxygen. The luciferase gene has recently been cloned and proposed as a reporter gene in procaryotic and eucaryotic cells. We present here a luciferase activity assay which relies on luminescence detection using a standard scintillation counter. This technique is simple, fast, inexpensive, and still very sensitive: as little as 0.02 pg (250,000 molecules) of enzyme is readily detected. The technique is optimized for the luciferase assay in mammalian cell lysates. Thus, the luciferase gene may become a very useful tool for gene regulation studies.     

Improved assay sensitivity of an engineered secreted Renilla luciferase.

We have previously reported the construction of a functional Renilla luciferase enzyme secreted by mammalian cells when fused to the signal peptide of human interleukin-2. The presence of three predicted cysteine residues in the amino acid sequence of Renilla luciferase suggested that its secreted form could contain oxidized sulfhydryls, which might impair enzyme activity. In this work, four secreted Renilla luciferase mutants were constructed to investigate this possibility: three luciferase mutants in which a different cysteine residue was replaced by an alanine residue, and one luciferase mutant in which all three cysteine residues were replaced by alanine residues. Simian cells were transfected with the genes encoding these mutant luciferases, as well as with the original gene construct, and cell culture media were assayed for bioluminescence activity. Only media containing a mutated luciferase with a cysteine to alanine substitution at position 152 in the preprotein showed a marked increase in bioluminescence activity when compared to media containing the original secreted Renilla luciferase. This increase in light emission was due in part to enhanced stability of the mutant enzyme. This new enzyme represents a significant improvement in the sensitivity of the secreted Renilla luciferase assay for monitoring gene expression.     

Oplophorus oxyluciferin and a model luciferin compound biologically active with Oplophorus luciferase.

The luciferin of the bioluminescent decapod shrimp, Oplophorus gracilorostris, was purified and studied with respect to u.v. spectrum, fluorescence spectrum, mass spectrum and luminescent cross-reaction with the enzyme luciferase of the bioluminescent ostracod, Cypridina hilgendorfii. On the basis of these results, an empirical formula C10H13N3O3 and an imidazo [1,2-a]pyrazin-3-one structure are proposed for luciferin. Of three model luciferin compounds, 3-hydroxy-2-methylimidazo[1,2-a]pyridine is biologically active with both Oplophorus and Cypridina luciferase, indicating that a pyrazine structure is not essential for biological activity with Cypridina luciferase.     

Bioluminescent monitoring enables observation of intracellular events in real time without cell and tissue destruction

Secreted reporter proteins provide monitoring of intracellular events in real time without cell destruction. To create human melanoma cell lines that enables noninvasive bioluminescent monitoring of metabolic activity, a comparison of the efficiency of isoforms and mutant variants of luciferase from the Metridia longa as secreted reporter proteins in the cells of human melanoma lines Mel IL was conducted. The MLM3 deletion mutant had the highest activity in the medium of two studied isoforms and two deletion mutants of secreted M. longa luciferase during the Mel IL melanoma cell transfection. It was established that optimization of the gene structure of the selected MLM3 variant for expression in human cells increases the level of bioluminescent activity in the Mel IL cells by almost an order of magnitude. A stable Mel IL melanoma cell line with constitutive expression of the humanized hMLM3 reporter gene was obtained and characterized. The linear range of identification of living cells by the hMLM3 reporter activity was more than three orders of magnitude with a sensitivity of detection of 10 cells.

     

Use of bacterial and firefly luciferases as reporter genes in DEAE-dextran-mediated transfection of mammalian cells.

The aim of this study was to compare three different luciferase genes by placing them in a single reporter vector and expressing them in the same mammalian cell type. The luciferase genes investigated were the luc genes from the fireflies Photinus pyralis (PP) and Luciola mingrelica (LM) and the lux AB5 gene, a translational fusion of the two subunits of the bacterial luciferase from Vibrio harveyi (VH). The chloramphenicol acetyltransferase (CAT) gene was also included in this study for comparison. The performances of the assay methods of the corresponding enzymes were evaluated using reference materials and the results of the expressed enzymes following transfection were calculated using calibration curves. All of the bioluminescent assays possess high reproducibility both within and between the batches (less than 15%). The comparison of the assay methods shows that firefly luciferases have the highest detection sensitivity (0.05 and 0.08 amol for PP and LM, respectively) whereas the VH bacterial luciferase has 5 amol and CAT 100 amol. On the other hand, the transfection of the various plasmids shows that the content of the expressed enzyme within the cells is much higher for CAT than for the other luciferase genes. VH luciferase is expressed at very low levels in mammalian cells due to the relatively high temperature of growing of the mammalian cells that seems to impair the correct folding of the active enzyme. PP and LM luciferases are both expressed at picomolar level but usually 10 to 70 times less in content with respect to CAT within the transfected cells. On the basis of these results the overall improvement in sensitivity related to the use of firefly luciferases as reporter genes in mammalian cells is about 30 to 50 times with respect to that of CAT.     

Expression of the gene encoding firefly luciferase in insect cells using a baculovirus vector

A cDNA encoding the firefly luciferase [Photinus luciferin: oxygen 4-oxidoreductase (decarboxylating, ATP-hydrolyzing), EC 1.13.12.7] was cloned downstream from the polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus and expressed in Spodoptera frugiperda clone-9 cells. Synthesis of luciferase (Luc) was accurately measured in insect cells growing in a 96-well plate, by a simple, rapid, nonradioactive, inexpensive and sensitive method based on fogging of x-ray film. Luc was produced in a coordinate fashion during virus infection. The Luc synthesized in insect cells was not secreted into the medium but was contained within the cell. Our findings suggest that Luc can be used as a superior reporter enzyme for molecular genetic analyses of baculovirus regulatory signals involved in high level expression of foreign genes, protein processing, targeting and stability in insect cells.     

Transfection efficiency of normal and cancer cell lines and monitoring of promoter activity by single‐cell bioluminescence imaging

The bioluminescence system (luciferase reporter assay system) is widely used to study gene expression, signal transduction and other cellular activities. Although transfection of reporter plasmid DNA to mammalian cell lines is an indispensable experimental step, the transfection efficiency of DNA varies among cell lines, and several cell lines are not suitable for this type of assay because of the low transfection efficiency. In this study, we confirm the transfection efficiency of reporter DNA to several cancer and normal cell lines after transient transfection by single‐cell imaging. Luminescence images could be obtained from living single cells after transient transfection, and the calculated transfection efficiency of this method was similar to that of the conventional reporter assay using a luminometer. We attempted to measure the activity of the Bip promoter under endoplasmic reticulum stress conditions using both high and low transfection efficiency cells for plasmid DNA at the single‐cell level, and observed activation of this promoter even in cells with the lowest transfection efficiency. These results show that bioluminescence imaging of single cells is a powerful tool for the analysis of gene expression based on a reporter assay using limited samples such as clinical specimens or cells from primary culture, and could provide additional information compared with the conventional assay. Copyright © 2013 John Wiley & Sons, Ltd.     

Kinetic analysis and modeling of firefly luciferase as a quantitative reporter gene in live mammalian cells

Firefly luciferase has proven to be a highly sensitive and quantitative reporter gene for studying gene delivery and regulation, and its recent use in live cells and organisms promises to further expand its utility. However, the intracellular behavior and properties of the enzyme are not well characterized. Specifically, information on the intracellular kinetics and stability of luciferase activity is necessary for real-time luminescence counts from live cells to be quantitatively meaningful. Here, we report a dynamic analysis of luciferase activity in the context of living mammalian cells. We have determined the relative light units measured in living cells to be proportional to that found in cell lysate. We have also calculated the K(m) of luciferase in living cells to be approximately 1 mM, a value much higher than the 10 microM found for pure enzyme in vitro. In addition, a 2-hour half-life of luciferase activity in live cells was measured in real time. Finally, we have modeled luciferase activity in live cells for the purposes of understanding and translating the luciferase signal into a more effective metric of gene expression and cell behavior.     

An improved CAT assay for promoter analysis in either transgenic mice or tissue culture cells.

We have developed an improved method for determining CAT activity directed by stably (transgenic mice) or transiently (tissue culture cell lines) introduced CAT reporter gene constructs. The procedure is based on the use of a new buffer system which considerably increases the stability of the CAT enzyme during the preparation of the crude cell extracts. When compared to other procedures, our method enables an increase of up to 100-fold in the sensitivity of the assay, depending on the transgenic tissue tested. Furthermore, a strong increase (up to 23-fold) was also observed with various promoter/CAT constructs transiently transfected in established tissue culture cell lines. This increase in sensitivity provides a significant reduction in the time required to perform the CAT assay when strong promoters are studied (from 18 to 1 hr) and is also very useful for the analysis of CAT gene expression driven by weak promoters.     

Development of a destabilized firefly luciferase enzyme for measurement of gene expression

Firefly luciferase is used widely as a reporter enzyme for studies of gene regulation and expression. The recent development of new technologies that combine luciferase reporter technology and digital imaging microscopy has enabled multiple measurements of gene expression in the same living cell. Although this approach has already provided new insights about expression dynamics, its future utility is limited by the three- to four-hour half-life of firefly luciferase in mammalian cells. Because of this, rapid increases or decreases in gene expression may not be detected, owing to the accumulation of residual luciferase. Accordingly, the goal of the present study was to develop a luciferase reporter with a reduced functional half-life. This was accomplished by adding a synthetic fragment to the firefly luciferase-coding sequence that encoded the proteolytic "PEST" signal from mouse ornithine decarboxylase. When placed under the control of estrogen response elements and expressed in human breast cancer T-47D cells, the modified luciferase protein (LUCODC-DA) displayed a functional half-life of 0.84 h compared to 3.68 h for the wild-type enzyme. As anticipated, the overall rate of photonic emissions in cells expressing the destabilized luciferase was about sevenfold lower than that of their wild-type counterparts, presumably because of the reduction of steady-state luciferase accumulation. Even so, the photonic activity derived from LUCODC-DA was still sufficient to enable real-time measurements of gene expression in single living cells.     

A real time Metridia luciferase based non-invasive reporter assay of mammalian cell viability and cytotoxicity via the β-actin promoter and enhancer

Secreted reporter molecules offer a means to evaluate biological processes in real time without the need to sacrifice samples at pre-determined endpoints. Here we have adapted the secreted bioluminescent reporter gene, Metridia luciferase, for use in a real-time viability assay for mammalian cells. The coding region of the marine copepod gene has been codon optimized for expression in human cells (hMLuc) and placed under the control of the human β-actin promoter and enhancer. Metridia luciferase activity of stably transfected cell models corresponded linearly with cell number over a 4-log dynamic range, detecting as few as 40 cells. When compared to standard endpoint viability assays, which measure the mitochondrial dehydrogenase reduction of tetrazolium salts, the hMLuc viability assay had a broader linear range of detection, was applicable to large tissue culture vessels, and allowed the same sample to be repeatedly measured over several days. Additional studies confirmed that MLuc activity was inhibited by serum, but demonstrated that assay activity remained linear and was measurable in the serum of mice bearing subcutaneous hMLuc-expressing tumors. In summary, these comparative studies demonstrate the value of humanized Metridia luciferase as an inexpensive and non-invasive method for analyzing viable cell number, growth, tumor volume, and therapeutic response in real time.     

SAMY, a novel mammalian reporter gene derived from Bacillus stearothermophilus alpha-amylase

The Bacillus stearothermophilus alpha-amylase (amyS) is a heat-stable monomeric exoenzyme which catalyses random hydrolysis of 1,4-alpha-glucosidic linkages in polyglucosans. The Bacillus alpha-amylase was engineered for use as an intracellular (AmyS(Delta S)) as well as a secreted reporter protein (SAMY; secreted alpha-amylase) in mammalian cells. The 5' end of amyS containing the prokaryotic secretion signal was either deleted (amyS(Delta S)) or replaced by a murine immunoglobulin secretion signal. SAMY was cloned under control of the cytomegalovirus promoter (P(CMV)) in a mammalian expression vector or the promoter of the human elongation factor 1 alpha (P(EF1 alpha)) in a lentiviral expression context. A variety of mammalian and human cell lines growing as monolayers, in suspension or as three-dimensional spheroids were transfected/transduced with SAMY- or amyS(Delta S)-encoding expression/lentiviral vectors and alpha-amylase activity was measured in cell lysates and culture supernatants. These experiments showed that SAMY and AmyS(Delta S) were either secreted or remained intracellular as highly sensitive reporter enzymes. SAMY expression and detection was fully compatible with established SEAP (human secreted alkaline phosphatase) and u-PA(LMW) (low molecular weight urokinase-type plasminogen activator) reporter systems and could be used to quantify expression of up to three independent genes in one culture supernatant.     

Induction of CAT gene expression on a plasmid vector (L factor) by retinoic acid in mouse embryonal carcinoma (F9) cells

We reported previously that composite DNA constructed from a mammalian plasmid (L factor) and foreign gene can be reestablished as a plasmid in mouse embryonal carcinoma (F9) cells after transfection and the plasmid-bearing F9 cells undergo normal in vitro differentiation in response to retinoic acid, an inducer for F9 cell differentiation. We constructed F9 cells bearing plasmidal L factor DNA in which a reporter (chloramphenicol acetyltransferase; CAT) gene was placed under the control of a differentiation-responsive viral (Moloney murine leukemia virus or simian virus 40) enhancer-promoter. When such plasmid-bearing cells were treated with retinoic acid, the CAT gene was inducibly expressed. These results indicate that mammalian gene expression can be studied with the plasmidal expression vector which is structurally dissociated from complex chromosomes.     

Real-time bioluminescence imaging of a protein secretory pathway in living mammalian cells using Gaussia luciferase

Using photon counting and charge-coupled device (CCD) cameras, we have applied the method of real-time bioluminescence imaging to investigate protein trafficking in mammalian cells. In the living cells of Chinese hamster ovary and PC12D cells, exocytotic secretion of protein and protein targeting on the cell surface were visualized using the secreted Gaussia luciferase (GLase) as a reporter protein in a minute. After incubation of the cells with luciferin (coelenterazine) for 10min, luciferin was imported into the cells and the vesicle transport network in the cells could be shown by luminescence images of GLase activity. Further, we demonstrate that GLase with a heterologous signal peptide sequence is targeted to the cell surface in neuronally differentiated PC12D cells and luminescence signals could be detected in a few seconds.