Visualization of the dynamics of gene expression in the living mouse

Reporter genes can monitor the status and activity of recombinant genomes in a diverse array of organisms, from bacteria and yeast to plants and animals. We have combined luciferase reporter genes with a conditional gene expression system based on regulatory elements from the lac operon of Escherichia coli to visualize the dynamics of gene expression in realtime in the living mouse. Using this technology, we have determined the rate of gene induction and repression, the level of target gene activity in response to different doses of inducer, and the schedule of induction during early embryogenesis of both the endogenous and the experimentally manipulated programs of mammalian gene expression associated with the HD/Hdh locus. The combination of in vivo imaging and lac regulation is a powerful tool for generating conditional transgenic mice that can be screened rapidly for optimal regulation and expression patterns, and for monitoring the induction and repression of regulated genes noninvasively in the living animal.     

A 'select and swap' strategy for the isolation of clones with tightly regulated transgenes.

Increasing numbers of biological problems are being addressed by genetic approaches that rely on inducible expression of transgenes. It is desirable that expression of such a transgene is tightly regulated, from close to zero expression in the 'off' state, to appreciable (at least physiological) expression in the 'on' state. Although there are many examples where tight regulation has been achieved, certain factors, including chromosomal position effects due to random integration of the transgene, often cause suboptimal inducibility and make the isolation of tightly regulated clones difficult and/or laborious. Here we describe a 'select and swap' strategy for the isolation, from a population of stable transfectants, of clones with tightly regulated transgenes. In this approach, a positively and negatively selectable, inducible marker gene is used to select for clones with optimal transgene regulation. After isolation of such clones, the marker gene is swapped with a linked gene of interest by the use of site-specific recombination. To test this strategy we introduced into human cells a plasmid with a tetracycline-inducible bacterial gpt gene linked to a promoterless luciferase gene, isolated clones with tight gpt expression and used the Cre/loxP site-specific recombination system to swap the gpt gene with the luciferase gene. We discuss ways for refining and developing the system and widening its applicability.     

Bioluminescent detection of endotoxin effects on HIV-1 LTR-driven transcription in vivo.

We investigated the effects of Gram-negative bacterial lipopolysaccharide (LPS) on luciferase expression in transgenic reporter mice in which luciferase expression is driven by the nuclear factor kappaB (NF-kappaB)-dependent portion of the human immunodeficiency virus-1 (HIV-1) long terminal repeat (HIV-1 LTR). Using these mice, we dissected the sources of luciferase activity at the organ level by (a) assessing luciferase activity in organ homogenates, (b) bioluminescence imaging in vivo, and (c) bioluminescence imaging of individual organs ex vivo. Luciferin dosage was a critical determinant of the magnitude of photon emission from these reporter mice. Photon emission increased at doses from 0.5-6 mg of luciferin given by intraperitoneal (IP) injection. The differential between basal and LPS-induced bioluminescence was maximal at 3-6 mg of luciferin. Luciferase expression was highly inducible in lungs, liver, spleen, and kidneys after a single IP injection of LPS, as assessed by luciferase activity measurements in organ homogenates. Luciferase activity was also induced in the forebrain by treatment with IP LPS. In contrast, aerosolized LPS produced a response localized to the lungs as assessed by both bioluminescence and ex vivo luciferase assay measurements. These studies demonstrate the utility of luciferase reporter mice for determining organ-specific gene expression in response to local and systemic stimuli.     

A bioluminescent transgenic mouse model: Real-time in vivo imaging of antioxidant EC-SOD gene expression and regulation by interferon gamma

Extracellular superoxide dismutase (EC-SOD) is the main antioxidant enzyme in the extracellular matrix. We developed transgenic mice to analyze the EC-SOD promoter activity in vivo in real time and to identify the important cis-elements flanking the 5′ region of the murine EC-SOD gene. Using this model, we demonstrated that luciferase reporter activity correlates closely with endogenous EC-SOD expression, although several interesting differences were also observed. Specifically, luciferase activity was detected at the highest levels in testes, aorta and perirenal fat. Reporter expression was regulated by interferon gamma, a finding that is in agreement with published endogenous EC-SOD gene expression studies. Thus, the 5′-flanking region of mouse EC-SOD gene is responsible, at least in part, for cell specific and inducible expression.     

In Vivo Bioluminescent Imaging of Influenza A Virus Infection and Characterization of Novel Cross-Protective Monoclonal Antibodies

Influenza A virus is a major human pathogen responsible for seasonal epidemics as well as pandemic outbreaks. Due to the continuing burden on human health, the need for new tools to study influenza virus pathogenesis as well as to evaluate new therapeutics is paramount. We report the development of a stable, replication-competent luciferase reporter influenza A virus that can be used for in vivo imaging of viral replication. This imaging is noninvasive and allows for the longitudinal monitoring of infection in living animals. We used this tool to characterize novel monoclonal antibodies that bind the conserved stalk domain of the viral hemagglutinin of H1 and H5 subtypes and protect mice from lethal disease. The use of luciferase reporter influenza viruses allows for new mechanistic studies to expand our knowledge of virus-induced disease and provides a new quantitative method to evaluate future antiviral therapies.     

Genetic modification of embryonic stem cells with VEGF enhances cell survival and improves cardiac function

Cardiac stem cell therapy remains hampered by acute donor cell death posttransplantation and the lack of reliable methods for tracking cell survival in vivo. We hypothesize that cells transfected with inducible vascular endothelial growth factor 165 (VEGF(165)) can improve their survival as monitored by novel molecular imaging techniques. Mouse embryonic stem (ES) cells were transfected with an inducible, bidirectional tetracycline (Bi-Tet) promoter driving VEGF(165) and renilla luciferase (Rluc). Addition of doxycycline induced Bi-Tet expression of VEGF(165) and Rluc significantly compared to baseline (p<0.05). Expression of VEGF(165) enhanced ES cell proliferation and inhibited apoptosis as determined by Annexin-V staining. For noninvasive imaging, ES cells were transduced with a double fusion (DF) reporter gene consisting of firefly luciferase and enhanced green fluorescence protein (Fluc-eGFP). There was a robust correlation between cell number and Fluc activity (R(2)=0.99). Analysis by immunostaining, histology, and RT-PCR confirmed that expression of Bi-Tet and DF systems did not affect ES cell self-renewal or pluripotency. ES cells were differentiated into beating embryoid bodies expressing cardiac markers such as troponin, Nkx2.5, and beta-MHC. Afterward, 5 x 10(5) cells obtained from these beating embryoid bodies or saline were injected into the myocardium of SV129 mice (n=36) following ligation of the left anterior descending (LAD) artery. Bioluminescence imaging (BLI) and echocardiography showed that VEGF(165) induction led to significant improvements in both transplanted cell survival and cardiac function (p<0.05). This is the first study to demonstrate imaging of embryonic stem cell-mediated gene therapy targeting cardiovascular disease. With further validation, this platform may have broad applications for current basic research and further clinical studies.     

Regulated gene expression from adenovirus vectors: a systematic comparison of various inducible systems

Positively and tightly regulated gene expression is essential for gene function and gene therapy research. The currently-used inducible gene expression systems include tetracycline (Tet-on and T-REx), ecdysone, antiprogestin and dimerizer-based systems. Adenovirus (Ad) vectors play an important role in gene function and gene therapy research for their various advantages over other vector systems. Previously, we reported the inferiority of the Tet-on system as an inducible gene expression system in the context of Ad vectors in comparison with the Tet-off system. In this study, to identify an optimal system for regulated gene expression from Ad vectors, we made a rigorous direct comparison of these five inducible gene expression systems in three cell lines using the luciferase reporter gene. The highest sensitivity to the respective inducer was that of the dimerizer system, followed by the antiprogestin system. The lowest basal expression and the highest induction factor were both characteristic of the dimerizer system. Furthermore, the dimerizer and T-REx systems exhibited much higher induced expression levels than the other three systems. The elucidation of the characteristic features of each system should provide important information for widespread and feasible application of these systems. Overall, these results suggest the most appropriate inducible gene expression system in the context of Ad vectors to be the dimerizer system.     

Noninvasive bioluminescence imaging of herpes simplex virus type 1 infection and therapy in living mice

Mouse models of herpes simplex virus type 1 (HSV-1) infection provide significant insights into viral and host genes that regulate disease pathogenesis, but conventional methods to determine the full extent of viral spread and replication typically require the sacrifice of infected animals. To develop a noninvasive method for detecting HSV-1 in living mice, we used a strain KOS HSV-1 recombinant that expresses firefly (Photinus pyralis) and Renilla (Renilla reniformis) luciferase reporter proteins and monitored infection with a cooled charge-coupled device camera. Viral infection in mouse footpads, peritoneal cavity, brain, and eyes could be detected by bioluminescence imaging of firefly luciferase. The activity of Renilla luciferase could be imaged after direct administration of substrate to infected eyes but not following the systemic delivery of substrate. The magnitude of bioluminescence from firefly luciferase measured in vivo correlated directly with input titers of recombinant virus used for infection. Treatment of infected mice with valacyclovir, a potent inhibitor of HSV-1 replication, produced dose-dependent decreases in firefly luciferase activity that correlated with changes in viral titers. These data demonstrate that bioluminescence imaging can be used for noninvasive, real-time monitoring of HSV-1 infection and therapy in living mice.     

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.     

Inducible gene expression with the Tet-on system in CD4+ T cells and thymocytes of mice

CD4+ T cells with their growing list of effector and regulatory subpopulations have vital functions within the immunohematopoietic system. We report here on the first mouse lines that allow temporally and quantitatively controlled expression of transgenes specifically in CD4+ thymocytes and T cells. These were constructed using the Tet-on system. The rtTA2(S)-M2 version of the reverse tetracycline-dependent transactivator was placed under control of all known CD4 regulatory elements. Reporter transgene expression in mice expressing these constructs is highly specific for CD4+ cells, is strictly dependent on the tetracycline derivative doxycycline, and can be regulated by up to five logs depending on the doxycycline concentration. Moreover, we demonstrate that these mice can be used for noninvasive in vivo imaging of a coexpressed luciferase reporter. These new mouse lines should be highly valuable for studying and manipulating numerous aspects of CD4+ T cell development, biology, and function.     

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.     

Micro-PET/CT monitoring of herpes thymidine kinase suicide gene therapy in a prostate cancer xenograft: the advantage of a cell-specific transcriptional targeting approach

Cancer gene therapy based on tissue-restricted expression of cytotoxic gene should achieve superior therapeutic index over an unrestricted method. This study compared the therapeutic effects of a highly augmented, prostate-specific gene expression method to a strong constitutive promoter-driven approach. Molecular imaging was coupled to gene therapy to ascertain real-time therapeutic activity. The imaging reporter gene (luciferase) and the cytotoxic gene (herpes simplex thymidine kinase) were delivered by adenoviral vectors injected directly into human prostate tumors grafted in SCID mice. Serial bioluminescence imaging, positron emission tomography, and computed tomography revealed restriction of gene expression to the tumors when prostate-specific vector was employed. In contrast, administration of constitutive active vector resulted in strong signals in the liver. Liver serology, tissue histology, and frail condition of animals confirmed liver toxicity suffered by the constitutive active cohorts, whereas the prostate-targeted group was unaffected. The extent of tumor killing was analyzed by apoptotic staining and human prostate marker (prostate-specific antigen). Overall, the augmented prostate-specific expression system was superior to the constitutive approach in safeguarding against systemic toxicity, while achieving effective tumor killing. Integrating noninvasive imaging into cytotoxic gene therapy will provide a useful strategy to monitor gene expression and therapeutic efficacy in future clinical protocols.     

Visualizing protein-protein interactions in living animals

A variety of techniques have been developed to analyze protein-protein interactions in vitro and in cultured cells. However, these methods do not determine how protein interactions affect and are regulated by physiologic and pathophysiologic conditions in living animals. This article describes methodology for detecting and quantifying protein interactions in living mice, using an inducible two-hybrid system developed for positron emission tomography (PET) imaging. We discuss the methods to establish stably transfected cells with components of the imaging system, create tumor xenografts, synthesize PET radiopharmaceuticals used to visualize the imaging reporter, perform microPET imaging, and analyze data from imaging studies. Development and application of technologies for molecular imaging of protein-protein interactions in vivo should enable researchers to investigate intrinsic binding specificities of proteins during normal development and disease progression as well as aid drug development through direct interrogation of molecular targets within intact animals.     

Let there be bioluminescence: development of a biophotonic imaging platform for in situ analyses of oral biofilms in animal models

In the current study, we describe a novel biophotonic imaging‐based reporter system that is particularly useful for the study of virulence in polymicrobial infections and interspecies interactions within animal models. A suite of luciferase enzymes was compared using three early colonizing species of the human oral flora (Streptococcus mutans, Streptococcus gordonii and Streptococcus sanguinis) to determine the utility of the different reporters for multiplexed imaging studies in vivo. Using the multiplex approach, we were able to track individual species within a dual‐species oral infection model in mice with both temporal and spatial resolution. We also demonstrate how biophotonic imaging of multiplexed luciferase reporters could be adapted for real‐time quantification of bacterial gene expression in situ. By creating an inducible dual‐luciferase expressing reporter strain of S. mutans, we were able to exogenously control and measure expression of nlmAB (encoding the bacteriocin mutacin IV) within mice to assess its importance for the persistence ability of S. mutans in the oral cavity. The imaging system described in the current study circumvents many of the inherent limitations of current animal model systems, which should now make it feasible to test hypotheses that were previously impractical to model.