Oxygen sensitivity of reporter genes: implications for preclinical imaging of tumor hypoxia

Reporter gene techniques have been applied toward studying the physiologic phenomena associated with tumor hypoxia, a negative prognostic indicator. The purpose of this study was to assess the potential adverse effects of hypoxic conditions on the effectiveness of four commonly used reporter genes: Renilla luciferase, monomeric red fluorescent protein, thymidine kinase, and lacZ. Tumor-forming A375 cells expressing a trifusion reporter consisting of Renilla luciferase, monomeric red fluorescent protein, and thymidine kinase were subjected to decreasing oxygen tensions and assayed for reporter expression and activity. A375 cells expressing beta-galactosidase were similarly exposed to hypoxia, with activity of the reporter monitored by cleavage of the fluorescent substrate 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)-beta-galactoside (DDAOG). Generation of signal in in vivo tumor models expressing bioluminescent or beta-galactosidase reporters were also examined over the course of hypoxic stresses, either by tumor clamping or the antivascular agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA). Our findings indicate that bioluminescent and fluorescent reporter activity are decreased under hypoxia despite minimal variations in protein production, whereas beta-galactosidase reporter activity per unit protein was unchanged. These results demonstrate that combining beta-galactosidase with the DDAOG optical probe may be a robust reporter system for the in vivo study of tumor hypoxia.     

Chimeric receptors as a tool for luminescent measurement of biological activities of steroid hormones

Some applications of chimeric cellular models are presented to study the biological activities of steroid hormones. We have used several chimeric constructs encoding the DNA binding domain of Gal4 yeast protein fused to the hormone binding domain of various steroid receptors (MR, PR, GR and ER). Interactions of these chimeric receptors with a 17-mer DNA sequence, specific for Gal-4, control expression of the firefly luciferase as a reporter gene. Stable transfected cell lines expressing the firefly luciferase under the control of different steroids were established and an efficient and easy sub-cloning was allowed with the help of an imaging system using a single-photon-counting camera. In the cell lines obtained, the bioluminescent response can be easily measured and thus used to measure specific biological activities of steroid agonists or antagonists. We observed that the responses are effector-concentration-dependent and their biological activities will be compared to those of native receptors.     

Toxin–antitoxin-stabilized reporter plasmids for biophotonic imaging of Group A streptococcus

Bioluminescence is a rapid and cost-efficient optical imaging technology that allows the detection of bacteria in real-time during disease development. Here, we report a novel strategy to generate a wide range of bioluminescent group A streptococcus (GAS) strains by using a toxin–antitoxin-stabilized plasmid. The bacterial luciferin–luciferase operon (lux) or the firefly luciferase gene (ffluc) was introduced into GAS via a stabilized plasmid. The FFluc reporter gave significantly stronger bioluminescent signals than the Lux reporter, and was generally more stable. Plasmid-based luciferase reporters could easily be introduced into a variety of GAS strains and the signals correlated linearly with viable cell counts. Co-expression of the streptococcal ω–ε–ζ toxin–antitoxin operon provided segregational stability in the absence of antibiotics for at least 17 passages in vitro and up to 7 days in a mouse infection model. In addition, genome-integrated reporter constructs were also generated by site-specific recombination, but were found to be technically more challenging. The quick and efficient generation of various M-type GAS strains expressing plasmid-based luciferase reporters with comparable and quantifiable bioluminescence signals allows for comparative analysis of different GAS strains in vitro and in vivo.     

用于活体成像的小鼠肺癌移植瘤模型的建立

本研究旨在建立可用于活体成像的小鼠肺癌移植瘤模型。利用脂质体将荧光素酶表达载体pGL4.17(luc2/neo)转染至人非小细胞肺癌细胞株A549,经G418筛选获得稳定表达荧光素酶的细胞克隆。根据体外生物发光情况及细胞的生长特性,从中挑选合适克隆,进行裸鼠皮下接种,SCID鼠尾静脉接种,建立肺癌移植瘤模型。利用活体成像系统监测肿瘤的生长转移情况,并用切片HE染色进一步验证小鼠模型移植瘤的原位成瘤和转移能力。实验结果表明:本研究成功地构建了可用于活体成像的小鼠肺癌移植瘤模型,模型稳定可靠、直观、灵敏,为肿瘤生长转移机制的研究及抗肿瘤药物的研发提供了重要工具。     

Continuous delivery of D-luciferin by implanted micro-osmotic pumps enables true real-time bioluminescence imaging of luciferase activity in vivo

Bioluminescence imaging (BLI) of luciferase reporters in small animal models offers an attractive approach to monitor regulation of gene expression, signal transduction, and protein-protein interactions, as well as following tumor progression, cell engraftment, infectious pathogens, and target-specific drug action. Conventional BLI can be repeated within the same animal after bolus reinjections of a bioluminescent substrate. However, intervals between image acquisitions are governed by substrate pharmacokinetics and excretion, therefore restricting temporal resolution of reinjection protocols to the order of hours, limiting analyses of processes in vivo with short time constants. To eliminate these constraints, we examined use of implanted micro-osmotic pumps for continuous, long-term delivery of bioluminescent substrates. Pump-assisted d-luciferin delivery enabled BLI for > or = 7 days from a variety of luciferase reporters. Pumps allowed direct repetitive imaging at < 5-minute intervals of the pharmacodynamics of proteasome- and IKK-inhibiting drugs in mice bearing tumors stably expressing ubiquitin-firefly luciferase or IkappaBalpha-firefly luciferase fusion reporters. Circadian oscillations in the olfactory bulbs of transgenic rats expressing firefly luciferase under the control of the period1 promoter also were temporally resolved over the course of several days. We conclude that implanted pumps provide reliable, prolonged substrate delivery for high temporal resolution BLI, traversing complications of repetitive substrate injections.     

Bioluminescent Leishmania expressing luciferase for rapid and high throughput screening of drugs acting on amastigote-harbouring macrophages and for quantitative real-time monitoring of parasitism features in living mice

In this study, we have established conditions for generating Leishmania amazonensis recombinants stably expressing the firefly luciferase gene. These parasites produced significant bioluminescent signals for both in vitro studies and the development of an in vivo model, allowing the course of the parasitism to be readily monitored in real time in the living animals such as laboratory mice. First, a model was established, using parasite-infected mouse macrophages for rapidly determining the activity of drugs against intracellular amastigotes. Results indicated that recombinant Leishmania can be reliably and confidently used to monitor compounds acting on intracellular amastigote-harbouring macrophages. Secondly, temporal analyses were performed following inoculation of metacyclic promastigotes into the ear dermis of BALB/c mice and the bioluminescent light transmitted through the tissue was imaged externally using a charge coupled device (CCD) camera. Bioluminescent signals, measured at the inoculation site and in the draining lymph node of mice containing these parasites correlated well with the more classical quantification of parasites. These assays prove that the real-time bioluminescent assay is not only sensitive but also more rapid than culture-base techniques allowing to monitor parasite-load before any clinical signs of leishmaniasis are detectable. In short, this luciferase imaging study is useful to monitor the efficacy of anti-leishmanial drugs on live cell culture and to trace leishmanial infection in animal models.     

Depot formation of doxycycline impairs Tet-regulated gene expression in vivo

The tetracycline (Tet) system is widely used for regulation of gene expression in vitro and in vivo. We constructed C57BL/6 transgenic mice (rtTA-CM2) with strong and ubiquitous reverse transactivator (rtTA2(S)-M2) gene expression. rtTA-CM2 mice were crossed to Tet-responsive reporter mice (LC-1) conditionally expressing the firefly luciferase (FLuc) gene under control of a Tet-responsive element, which allowed sensitive quantification of the transactivator activity by bioluminescent imaging. Following doxycycline (dox) application, up to 10(5)-fold increase in BL signal was measured. rtTA activity was inducible in most analyzed organs. After dox withdrawal the BL signal decreased significantly but did not disappear completely, most likely due to a dox depot formation in vivo. The residual dox was sufficient to partly down-regulate a Tet-off controlled oncogene in a tumor transplantation experiment, resulting in reduced tumor growth. rtTA-CM2 mice may be a useful tool to analyze the function of genes in various organs but also reveal that down-regulation of gene expression is not complete.     

In vivo imaging of retinoic acid receptor activity using a sodium/iodide symporter and luciferase dual imaging reporter gene

Retinoic acids are natural derivatives of vitamin A, and play important roles in modulating tumor cell growth by regulating differentiation, thus suggesting the potential use of these derivatives in cancer therapy and prevention. To visualize the intranuclear responses of functional retinoic acid receptors, we have developed a dual-imaging reporter gene system based on the use of sodium/iodide symporter (NIS) and luciferase in cancer cell lines. NIS and luciferase genes were linked with an internal ribosome entry site, and placed under the control of an artificial cis-acting retinoic acid responsive element (pRARE/NL). After retinoic acid treatment, I-125 uptake by pRARE/NL transfected cells was found to have increased by up to about five times that of nontreated cells. The bioluminescence intensity of pRARE/NL transfected cells showed dose-dependency. In vivo luciferase images showed higher intensity in retinoic acid treated SK-RARE/NL tumors, and scintigraphic images of SK-RARE/NL tumors showed increased Tc-99m uptake after retinoic acid treatment. The NIS/luciferase imaging reporter system was sufficiently sensitive to allow the visualization of intranuclear retinoic acid receptor activity. This cis-enhancer imaging reporter system may be useful in vitro and in vivo for the evaluation of retinoic acid responses in such areas as cellular differentiation and chemoprevention.     

利用萤光素酶标记的肿瘤细胞建立肝癌原位移植模型及其活体成像

目的：利用生物发光成像技术非侵入性地监测活体裸鼠原位肝癌发展过程。方法：将包含有萤火虫萤光素酶基因的pCI-neo-Luc载体转染人肝癌HepG2细胞系,筛选获得具有高萤光素酶活性的细胞克隆;利用流式细胞仪对萤光素酶表达的稳定性进行初步研究,并分析细胞的生物发光情况;持续表达萤光素酶的肿瘤细胞培养扩增后被植入裸鼠皮下,2周后以形成的异体瘤作为供体瘤,进行肝脏原位移植手术;对建立的肝癌原位移植模型,用影像学资料显示肿瘤部位,用IVIS成像系统动态监测肿瘤生长情况。结果：体外影像的结果显示,表达萤光素酶细胞的数量与发光强度呈正相关;活体成像的结果显示。成功地建立了萤光素酶标记的原位肝癌动物模型。结论：生物发光成像可以监测活体内肝癌演进过程,为抗肿瘤药物的筛选和评价提供了新的手段和工具。     

Bioluminescent Aspergillus fumigatus, a New Tool for Drug Efficiency Testing and In Vivo Monitoring of Invasive Aspergillosis

Aspergillus fumigatus is the main cause of invasive aspergillosis in immunocompromised patients, and only a limited number of drugs for treatment are available. A screening method for new antifungal compounds is urgently required, preferably an approach suitable for in vitro and in vivo studies. Bioluminescence imaging is a powerful tool to study the temporal and spatial resolutions of the infection and the effectiveness of antifungal drugs. Here, we describe the construction of a bioluminescent A. fumigatus strain by fusing the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene from A. fumigatus with the luciferase gene from Photinus pyralis to control the expression of the bioluminescent reporter. A. fumigatus transformed with this construct revealed high bioluminescence under all tested growth conditions. Furthermore, light emission correlated with the number of conidia used for inoculation and with the biomass formed after different incubation times. The bioluminescent strains were suitable to study the effectiveness of antifungals in vitro by several independent methods, including the determination of light emission with a microplate reader and the direct visualization of light emission with an IVIS 100 system. Moreover, when glucocorticoid-treated immunosuppressed mice were infected with a bioluminescent strain, light emission was detected from infected lungs, allowing the visualization of the progression of invasive aspergillosis. Therefore, this new bioluminescence tool is suitable to study the in vitro effectiveness of drugs and the disease development, localization, and burden of fungi within tissues and may also provide a powerful tool to study the effectiveness of antifungals in vivo.     

In vivo testing of Renilla luciferase substrate analogs in an orthotopic murine model of human glioblastoma

In vivo bioluminescent imaging using cells expressing Renilla luciferase is becoming increasingly common. Hindrances to the more widespread use of Renilla luciferase are the high autoluminescence of its natural substrate, coelenterazine, in plasma, the relatively high absorbance by tissue of the light emitted by the enzyme-substrate reaction; rapid clearance of the substrate; and significant cost. These factors, save for the cost, which has its own limiting effect on use, can combine to reduce the sensitivity of in vivo assays utilizing this reporter system, and methods of increasing light output or decreasing autoluminescence could be of great benefit. A number of analogs of coelenterazine are being investigated may accomplish one or both of these goals. In this study that we report on the testing of two new substrate analogs, EnduRen and ViViren, manufactured by Promega Corporation, in an orthotopic murine model of human glioblastoma expressing Renilla luciferase. We have tested these analogs in this cell line both in vitro and in vivo, and find that the substrate viviren results in significantly greater light output than the natural substrate or the other analog EnduRen. This new substrate could be valuable for studies where greater sensitivity is important.     

Comparison of noninvasive fluorescent and bioluminescent small animal optical imaging

Optical imaging is a modality that is cost-effective, rapid, easy to use, and can be readily applied to studying disease processes and biology in vivo. For this study, we used a green fluorescent protein (GFP)- and luciferase-expressing mouse tumor model to compare and contrast the quantitative and qualitative capabilities of a fluorescent reporter gene (GFP) and a bioluminescent reporter gene (luciferase). We describe the relationship between tumor volume, tumor mass, and bioluminescent/fluorescent intensity for both GFP and luciferase. Bioluminescent luciferase imaging was shown to be more sensitive than fluorescent GFP imaging. Luciferase-expressing tumors were detected as early as 1 day after tumor cell inoculation, whereas GFP-expressing tumors were not detected until 7 days later. Both bioluminescent and fluorescent intensity correlated significantly and linearly with tumor volume and tumor weight, as measured by caliper. Compared to bioluminescent imaging, fluorescent imaging does not require the injection of a substrate and may be appropriate for applications where sensitivity is not as critical. Knowing the relative strengths of each imaging modality will be important in guiding the decision to use fluorescence or bioluminescence.     

Prostaglandin F2alpha amplifies tumor necrosis factor-alpha promoter activity by the FPB prostanoid receptor

This study examines the regulation of tumor necrosis factor-alpha (TNF-alpha) promoter activity by prostaglandin F2alpha ( PGF2alpha ) in HEK cells stably expressing either the FPA or FPB prostanoid receptors. Cells were transiently transfected with a luciferase reporter plasmid under the control of a TNF-alpha promoter and luciferase activity was measured. In the absence of PGF2alpha basal TNF-alpha reporter gene activity is elevated in FPB cells as compared with FPA cells. This elevated basal activity is blocked by pretreatment with a Rho inhibitor, but not by pretreatment with an inhibitor of protein kinase C (PKC). TNF-alpha reporter activity in FPB cells is stimulated by PGF2alpha and this is decreased by pretreatment with a chelator of intracellular calcium or by a gap junction inhibitor. In FPB cells pretreatment with a Rho inhibitor combined with either a calcium chelator or a gap junction inhibitor decreases both basal and PGF2alpha stimulated TNF-alpha reporter activity. Interestingly post-treatment of FPB cells with an inhibitor of PKC decreased PGF2alpha stimulated TNF-alpha reporter gene activity even though pretreatment did not. It, therefore, appears that PGF2alpha stimulated TNF-alpha reporter activity in FPB cells is amplified by a Rho-dependent mechanism involving calcium, gap junctions, and PKC. These findings may help in understanding the function of the FPB isoform in the corpus luteum.     

The sea pansy Renilla reniformis luciferase serves as a sensitive bioluminescent reporter for differential gene expression in Candida albicans.

The infectious yeast Candida albicans progresses through two developmental programs which involve differential gene expression, the bud-hypha transition and high-frequency phenotypic switching. To understand how differentially expressed genes are regulated in this organism, the promoters of phase-specific genes must be functionally characterized, and a bioluminescent reporter system would facilitate such characterization. However, C. albicans has adopted a nontraditional codon strategy that involves a tRNA with a CAG anticodon to decode the codon CUG as serine rather than leucine. Since the luciferase gene of the sea pansy Renilla reinformis contains no CUGs, we have used it to develop a highly sensitive bioluminescent reporter system for C. albicans. When fused to the galactose-inducible promoter of GAL1, luciferase activity is inducible; when fused to the constitutive EF1 alpha 2 promoter, luciferase activity is constitutive; and when fused to the promoter of the white-phase-specific gene WH11 or the opaque-phase-specific gene OP4, luciferase activity is phase specific. The Renilla luciferase system can, therefore, be used as a bioluminescent reporter to analyze the strength and developmental regulation of C. albicans promoters.     

Development of a luciferase reporter gene, luxCt, for Chlamydomonas reinhardtii chloroplast

Luciferase reporter genes have been successfully used in a variety of organisms to examine gene expression in living cells, but are yet to be successfully developed for use in chloroplast. Green fluorescent protein (gfp) has been used as a reporter of chloroplast gene expression, but because of high auto-fluorescence, very high levels of GFP accumulation are required for visualization in vivo. We have developed a luciferase reporter for chloroplast by synthesizing the two-subunit bacterial luciferase (lux)AB, as a single fusion protein in Chlamydomonas reinhardtii chloroplast codon bias. We expressed a chloroplast luciferase gene, luxCt, in C. reinhardtii chloroplasts under the control of the ATPase alpha subunit (atpA) or psbA promoter and 5' untranslated regions (UTRs) and the rubisco large subunit (rbcL) 3' UTR. We show that luxCt is a sensitive reporter of chloroplast gene expression, and that luciferase activity can be measured in vivo using a charge coupled device (CCD) camera or in vitro using a luminometer. We further demonstrate that luxCt protein accumulation, as measured by Western blot analysis, is proportional to luminescence, as determined both in vivo and in vitro, and that luxCt is capable of reporting changes in chloroplast gene expression during a dark to light shift. These data demonstrate the utility of the luxCt gene as a versatile and sensitive reporter of chloroplast gene expression in living cells.     

A guanosine 3',5'-cyclic monophosphate (cGMP) reporter system based on the G-kinase/CREB/CRE signal transduction pathway

Guanylate cyclases constitute a gene family of enzymes that synthesize the second messenger guanosine 3′,5′-cyclic monophosphate (cGMP) and play important roles in diverse physiological functions. Here we report a novel, simple and highly sensitive method for measurement intracellular cGMP concentrations using a cAMP-responsive element (CRE) and cGMP-dependent protein kinase (cGK). Transient transfection of the CRE reporter plasmid, encoding a luciferase reporter gene under the control of a modified promoter containing a CRE, and a cGK expression vector into HEK293 cells followed by treatment with 8-bromo-cGMP showed a dose dependent increase in luciferase activity. Moreover, HEK293 cells expressing GC-A or GC-B natriuretic peptide receptors and harboring this reporter system responded to specific ligands in a dose dependent manner. Our results indicate that this reporter gene method enables high throughput screening of receptor-type GC selective agonists in the treatment of cardiovascular diseases and homeostatic dysfunctions.     

Use of a dual firefly and Renilla luciferase reporter gene assay to simultaneously determine drug selectivity at human corticotrophin releasing hormone 1 and 2 receptors

The aim of this study was to investigate and validate the use of a dual glow-signal luciferase reporter gene assay to simultaneously evaluate drug activity at two different seven-transmembrane receptor subtypes. Stable cell lines (CHO) transfected with either human corticotrophin releasing hormone 1 (hCRH1) receptors and a firefly luciferase reporter gene or hCRH2 and a Renilla luciferase reporter gene were created to provide different luciferase readouts for CRH1 and CRH2 receptors, respectively. Cells were combined for stimulation and measurement of luciferase luminescence in a 96-well plate format assay. The nonselective CRH agonists rat/human CRH and sauvagine caused concentration-dependent increases in luminescence via activation of CRH1 (firefly luciferase; pEC50 = 8.40 +/- 0.06 and 8.39 +/- 0.08, respectively, n = 8) and CRH2 (Renilla luciferase; pEC50 = 8.89 +/- 0.14 and 8.92 +/- 0.13, respectively, n = 8) receptors. The nonselective CRH antagonist astressin blocked these agonist-induced increases in luciferase at both CRH1 and CRH2 receptors. The selective CRH1 antagonist CP154,526 blocked r/hCRH- and sauvagine-induced increases in luciferase at CRH1 receptors only. These data report the expected pharmacology for CRH1 and CRH2 receptors. This assay enabled two receptor subtypes to be studied simultaneously in the same 96-well plate and generated robust data with low variability. It has the potential advantage of significant time and cost savings, with application to both basic research and compound screening.