Visualization of GFP-expressing tumors and metastasis in vivo

We have developed mouse models of metastatic cancer with genetically fluorescent tumors that can be imaged in fresh tissue, in situ, as well as externally. To achieve this capability, we have transduced the green fluorescent protein (GFP) gene, cloned from the bioluminescent jellyfish Aequorea victoria, into a series of human and rodent cancer cell lines that were selected in vitro to stably express GFP in vivo after transplantation to metastatic rodent models. Techniques were also developed for transduction of tumors by GFP in vivo. With this fluorescent tool, we detected and visualized for the first time tumors and metastasis in fresh viable tissue or in situ in host organs down to the single-cell level. GFP tumors on the colon, prostate, breast, brain, liver, lymph nodes, lung, pancreas, bone, and other organs can also be visualized externally, transcutaneously by quantitative whole-body fluorescence optical imaging. Real-time tumor and metastatic growth and angiogenesis and inhibition by representative drugs can be imaged and quantified for rapid antitumor, antimetastatic, and antiangiogenesis drug screening. The GFP-transfected tumor cells enabled a fundamental advance in the visualization of tumor growth and metastasis in real time in vivo.     

Bioluminescent imaging: applications to cancerology and endocrinology

Our laboratory has been using various bioluminescent imaging systems for more than 20 years to visualize and quantify bioluminescent and chemiluminescent reactions. This equipment allowed us to establish numerous cell lines expressing bioluminescent reporter genes to study the mechanism of action of nuclear receptors. Cells expressing the luciferase gene under the control of a constitutive promoter were used to follow in vivo proliferation of cancer cells. Intensities of in vitro and in vivo bioluminescent signals were compared and the conditions of bioluminescent reaction measurements were determined. These bioluminescent models are new tools for evaluating cancer treatment efficiencies and the role of hormone receptors in invasion. Cells expressing the luciferase gene under the control of hormones are used as in vivo biosensors for studying analog bioavailabilities and in vivo response kinetics. They are complementary models to in vitro models that have been developed in our laboratory for several years. In the future, targeting reporter gene (luciferase and GFP) expression to specific tissues should allow the detailed localisation of the action of nuclear receptor ligands.     

Optimisations and Challenges Involved in the Creation of Various Bioluminescent and Fluorescent Influenza A Virus Strains for In Vitro and In Vivo Applications

Bioluminescent and fluorescent influenza A viruses offer new opportunities to study influenza virus replication, tropism and pathogenesis. To date, several influenza A reporter viruses have been described. These strategies typically focused on a single reporter gene (either bioluminescent or fluorescent) in a single virus backbone. However, whilst bioluminescence is suited to in vivo imaging, fluorescent viruses are more appropriate for microscopy. Therefore, the idea l reporter virus varies depending on the experiment in question, and it is important that any reporter virus strategy can be adapted accordingly. Herein, a strategy was developed to create five different reporter viruses in a single virus backbone. Specifically, enhanced green fluorescent protein (eGFP), far-red fluorescent protein (fRFP), near-infrared fluorescent protein (iRFP), Gaussia luciferase (gLUC) and firefly luciferase (fLUC) were inserted into the PA gene segment of A/PR/8/34 (H1N1). This study provides a comprehensive characterisation of the effects of different reporter genes on influenza virus replication and reporter activity. In vivo reporter gene expression, in lung tissues, was only detected for eGFP, fRFP and gLUC expressing viruses. In vitro, the eGFP-expressing virus displayed the best reporter stability and could be used for correlative light electron microscopy (CLEM). This strategy was then used to create eGFP-expressing viruses consisting entirely of pandemic H1N1, highly pathogenic avian influenza (HPAI) H5N1 and H7N9. The HPAI H5N1 eGFP-expressing virus infected mice and reporter gene expression was detected, in lung tissues, in vivo. Thus, this study provides new tools and insights for the creation of bioluminescent and fluorescent influenza A reporter viruses.     

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.     

Evaluation of the reversal of multidrug resistance by MDR1 ribonucleic acid interference in a human colon cancer model using a Renilla luciferase reporter gene and coelenterazine

The reversal effect of multidrug resistance (MDR1) gene expression by adenoviral vector-mediated MDR1 ribonucleic acid interference was assessed in a human colon cancer animal model using bioluminescent imaging with Renilla luciferase (Rluc) gene and coelenterazine, a substrate for Rluc or MDR1 gene expression. A fluorescent microscopic examination demonstrated an increased green fluorescent protein signal in Ad-shMDR1- (recombinant adenovirus that coexpressed MDR1 small hairpin ribonucleic acid [shRNA] and green fluorescent protein) infected HCT-15/Rluc cells in a virus dose-dependent manner. Concurrently, with an increasing administered virus dose (0, 15, 30, 60, and 120 multiplicity of infection), Rluc activity was significantly increased in Ad-shMDR1-infected HCT-15/Rluc cells in a virus dose-dependent manner. In vivo bioluminescent imaging showed about 7.5-fold higher signal intensity in Ad-shMDR1-infected tumors than in control tumors (p < .05). Immunohistologic analysis demonstrated marked reduction of P-glycoprotein expression in infected tumor but not in control tumor. In conclusion, the reversal of MDR1 gene expression by MDR1 shRNA was successfully evaluated by bioluminescence imaging with Rluc activity using an in vivo animal model with a multidrug resistance cancer xenograft.     

An Optimized Triple Modality Reporter for Quantitative In Vivo Tumor Imaging and Therapy Evaluation

We present an optimized triple modality reporter construct combining a far-red fluorescent protein (E2-Crimson), enhanced firefly luciferase enzyme (Luc2), and truncated wild type herpes simplex virus I thymidine kinase (wttk) that allows for sensitive, long-term tracking of tumor growth in vivo by fluorescence, bioluminescence, and positron emission tomography. Two human cancer cell lines (MDA-MB-231 breast cancer and HT-1080 fibrosarcoma cancer) were successfully transduced to express this triple modality reporter. Fluorescence and bioluminescence imaging of the triple modality reporter were used to accurately quantify the therapeutic responses of MDA-MB-231 tumors to the chemotherapeutic agent monomethyl auristatin E in vivo in athymic nude mice. Positive correlation was observed between the fluorescence and bioluminescence signals, and these signals were also positively correlated with the ex vivo tumor weights. This is the first reported use of both fluorescence and bioluminescence signals from a multi-modality reporter construct to measure drug efficacy in vivo.     

Loss of a reporter gene for green fluorescent protein during tumor progression suggests the recruitment of host cells in rats with experimentally induced colon cancer

The interactions between a host's normal cells and tumor cells appear to be of significant importance during the development of tumors. In the present study, we examined this issue using a cancer model in vivo in which tumor cells were tagged with a reporter gene for green fluorescent protein (GFP). We used a model of colon cancer in immunocompetent rats, which were given a subcutaneous injection of tumor cells that had been transfected with a gene for GFP. We found that the number of fluorescent cells decreased with the progression of the primary tumors and that lymph node and lung metastases were never macroscopically fluorescent. No GFP-encoding sequences were detected by PCR in many of the long-term primary tumors, in most lymph node metastases (86%) and in all lung metastases, whereas the detection of mutated k-ras, which identified such cells as tumor cells, was always positive. To explain these findings, we present a brief review of the literature and postulate that tumor growth did not occur exclusively as a result of the division of the injected cells, but also involved recruitment of host cells.     

人胰腺癌细胞再增殖体外模型的建立

目的:细胞再增殖是导致胰腺癌放化疗失败的主要原因之一,但缺乏合适的用于研究胰腺癌再增殖的细胞模型。本研究拟建立简便、实用的胰腺癌细胞再增殖体外模型。方法:表达绿色荧光蛋白-荧光素酶(GFP-Luc)的慢病毒感染人胰腺癌细胞,经嘌呤霉素筛选,用荧光显微镜和流式细胞仪观察双标记细胞GFP表达情况,用生物成像检测双标记细胞Luc活性及分析细胞数量与Luc活性之间的关系。以X-线照射胰腺癌细胞制备饲养细胞,以相应的双标记肿瘤细胞为报告细胞进行共培养。对共培养细胞进行荧光显微镜观察和生物成像,以判断饲养细胞对报告细胞的生长促进作用。结果:通过表达GFP-Luc的慢病毒感染获得双标记人胰腺癌细胞,经荧光显微术、流式细胞术和生物成像术证实这些双标记的人胰腺癌细胞能有效地表达GFP和Luc活性,可作为报告细胞用于建立人胰腺癌再增殖细胞模型。将经X-线照射的饲养细胞和相应的报告细胞共培养,经荧光显微镜观察和生物成像分析,结果显示X-线照射过的饲养细胞对报告细胞的生长具有显著的促进作用。结论:成功建立了简便、实用的人胰腺癌再增殖体外模型,该模型能很好地模拟人体内胰腺癌细胞再增殖过程,为进一步研究胰腺癌细胞再增殖的分子机制提供了新的技术手段。     

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.     

Optimized expression of dual reporter genes in transient transfection of purified Toxoplasma gondii using different promoters

Fluorescent protein and luciferase genes are valuable reporter genes and have been widely used for noninvasive monitoring of gene expression in living tissues and cells. We tested expression of the dual reporter genes in transient transfection of purified Toxoplasma gondii tachyzoites. Two copies of the enhanced yellow fluorescent protein (EYFP) gene were put under the control of 3 representative T.?gondii promoters (GRA1, SAG1, and DHFR). Fluorescence from each EYFP reporter was significantly higher than that from a green fluorescent protein (GFP) reporter. The GRA1-EYFP reporter gave the highest fluorescence. Although both fluorescence and luciferase were expressed in the dual reporter system, the luciferase reporter was more efficient than either the EYFP or GFP reporters, and it required fewer parasites to be successfully used.     

Evaluation and Comparison of the GUS, LUC and GFP Reporter System for Gene Expression Studies in Plants

Abstract: The detailed analysis of the expression pattern of a plant gene can give important clues about its function in plant development, cell differentiation and defence reactions. Gene expression studies have been greatly facilitated by the employment of proteins like β-glucuronidase (GUS), green fluorescent protein (GFP), and firefly luciferase (LUC) as reporters of gene activity. The application of reporter genes in plants, specifically in the field of gene expression studies, has expanded over the years from a mere tool to quantify (trans) gene expression in tissue samples, to real-time imaging of in planta promoter dynamics. To correctly interpret the activity that is given by each reporter, it is important to have a good understanding of the intrinsic properties of the different reporter proteins. Here we discuss those properties of GUS, LUC and GFP that are of interest in gene expression studies.     

Formulation of photon diffusion from spherical bioluminescent sources in an infinite homogeneous medium

Background  

The bioluminescent enzyme firefly luciferase (Luc) or variants of green fluorescent protein (GFP) in transformed cells can be effectively used to reveal molecular and cellular features of neoplasia in vivo. Tumor cell growth and regression in response to various therapies can be evaluated by using bioluminescent imaging. In bioluminescent imaging, light propagates in highly scattering tissue, and the diffusion approximation is sufficiently accurate to predict the imaging signal around the biological tissue. The numerical solutions to the diffusion equation take large amounts of computational time, and the studies for its analytic solutions have attracted more attention in biomedical engineering applications.     

The color of mice: in the light of GFP-variant reporters

The mouse currently represents the premier model organism for mammalian genetic studies. Over the past decade the production of targeted and transgenic lines of mice has become commonplace, with current technology allowing the creation of mutations at base pair resolution. Such genome modifications are becoming increasingly elaborate and often incorporate gene-based reporters for tagging different cellular populations. Until recently, lacZ, the bacterial beta-galactosidase gene has been the marker of choice for most studies in the mouse. However, over the past 3 years another valuable reporter has emerged, and its attractiveness is reflected by an explosion in its use in mice. Green fluorescent protein (GFP), a novel autofluorescent genetic reporter derived from the bioluminescent jellyfish Aequorea victoria, currently represents a unique alternative to other gene-based reporters in that its visualization is non-invasive and so can be monitored in real-time in vitro or in vivo. It has the added advantage that it can be quantified by, for example, flow cytometry, confocal microscopy, and fluorometric assays. Several mutants of the original wild-type GFP gene that improve thermostability and fluorescence have been engineered. Enhanced GFP is one such variant, which has gained popularity for use in transgenic or targeted mice. Moreover, various GFP spectral variants have also been developed, and two of these novel color variants, enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP), can also be used in mice. Since the spectral profiles of the ECFP and EYFP color variants are distinct and non-overlapping, these two reporters can be co-visualized, and are therefore ideal for in vivo double-labeling or fluorescent energy transfer analyses. The use of GFP and its color variants as reporters provides an unprecedented level of sophistication and represents the next step in mouse genome engineering technology by opening up the possibility of combinatorial non-invasive reporter usage within a single animal.     

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.     

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.     

Codon-optimized Luciola italica luciferase variants for mammalian gene expression in culture and in vivo

Luciferases have proven to be useful tools in advancing our understanding of biologic processes. Having a multitude of bioluminescent reporters with different properties is highly desirable. We characterized codon-optimized thermostable green- and red-emitting luciferase variants from the Italian firefly Luciola italica for mammalian gene expression in culture and in vivo. Using lentivirus vectors to deliver and stably express these luciferases in mammalian cells, we showed that both variants displayed similar levels of activity and protein half-lives as well as similar light emission kinetics and higher stability compared to the North American firefly luciferase. Further, we characterized the red-shifted variant for in vivo bioluminescence imaging. Intramuscular injection of tumor cells stably expressing this variant into nude mice yielded a robust luciferase activity. Light emission peaked at 10 minutes post-d-luciferin injection and retained > 60% of signal at 1 hour. Similarly, luciferase activity from intracranially injected glioma cells expressing the red-shifted variant was readily detected and used as a marker to monitor tumor growth over time. Overall, our characterization of these codon-optimized luciferases lays the groundwork for their further use as bioluminescent reporters in mammalian cells.     

含分泌型萤光素酶和绿色荧光蛋白双报告基因的慢病毒载体的制备与表达分析

目的:制备含分泌型萤光素酶和绿色荧光蛋白双报告基因的慢病毒载体,为慢病毒载体的进一步广泛应用奠定基础。方法:克隆构建含分泌型萤光素酶和绿色荧光蛋白双报告基因的转基因载体pCS-gluc-2A-eGFP,酶切与序列分析鉴定其正确后,与包装质粒pCMVHR’Δ8.2、包膜质粒pVSV-G共转染293FT细胞,获得含分泌型萤光素酶和绿色荧光蛋白双报告基因的重组慢病毒载体;重组慢病毒载体感染A549、Huh7细胞后,用荧光显微镜直接观察报告基因GFP的表达,或取细胞上清实时检测分泌型萤光素酶的表达。结果:制备了含双报告基因的重组慢病毒载体,感染细胞后可以活体观察绿色荧光蛋白的表达,也可以快速灵敏地检测到分泌型萤光素酶的表达。结论:所获含分泌型萤光素酶和绿色荧光蛋白双报告基因的重组慢病毒载体感染效率高,表达易于活体实时检测,灵敏度高。本研究为慢病毒载体的广泛应用奠定了基础。     

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies ^1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays ⁴. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor ^4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells ^7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.     

Detection of Mercury in Aquatic Environments Using EPRE Reporter Zebrafish

It has been proposed that transgenic zebrafish could be designed to detect low levels of chemical contaminants that cause oxidative stress in aquatic environments, such as heavy metals or pesticides. In this paper, we describe such a transgenic zebrafish that produces a luciferase–green fluorescent protein (LUC–GFP) fusion protein under conditions of oxidative stress. The reporter gene expression is under the regulation of the electrophile responsive element (EPRE), which activates gene expression in response to oxidative stressors. The GFP component of this fusion protein allows us to visually detect reporter gene activity in live animals to determine if activity is localized to a particular tissue. The luciferase component is capable of returning a quantitative assessment of reporter gene activity that allows us to determine if reporter gene activity is directly correlated to the concentration of the chemical inducer. We have tested this reporter construct in both transient and stable transgenic fish after exposure to a range of HgCl₂ concentrations. GFP expression from the EPRE–LUC–GFP construct was inducible in transient assays but was below the limit of detection in stable lines. In contrast, we observed inducible luciferase activity in both transient assays and stable lines treated with HgCl₂. We conclude that the EPRE is capable of driving reporter gene expression in a whole animal assay under conditions of oxidative stress. Furthermore, expression was induced at HgCl₂ concentrations that do not result in obvious morphological defects, making this approach useful for the detection of low levels of oxidative contaminants in aquatic environments.