Bioluminescent indicators for in vivo measurements of gene expression

Trends in Biotechnology Vol. 20 No. 8 (Suppl.), 2002

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Bioluminescent indicators for in vivo measurements of gene expression Caitlin E. O’Connell-Rodwell, Stacy M. Burns, Michael H. Bachmann and Christopher H. Contag Recent developments in in vivo imaging using optical, radionuclide and paramagnetic reporter probes now enables continuous measurements of gene expression in living animals. In vivo bioluminescence imaging (BLI) is a sensitive, versatile and accessible imaging strategy that has been applied to a variety of small-animal models of human biology and disease. We discuss current strategies in BLI and the potential of combining BLI with other in vivo and ex vivo techniques. BLI will have a significant role in in vivo cellular and molecular imaging, a field that will help reveal the molecular basis of biology and disease. Ultra-sensitive and high-throughput quantitative in vivo analyses of gene expression and protein function have been made possible by the recent developments in the emerging field of in vivo cellular and molecular imaging. Several imaging systems use optical reporters that consist of either fluorescent proteins [1], bioluminescent proteins or a combination of these [2] to study gene expression. Other modalities, such as single photon emission computed tomography (SPECT) and positron emission tomography (PET), use radionuclides and reporter genes [3], and magnetic resonance imaging (MRI) relies on magnetic reporters or spectroscopic signatures to assess function [4]. Monitoring in vivo processes continuously and noninvasively in living animals using optical methods requires genes that encode reporters, such as luciferase, or fluorescent proteins coupled to genes of interest or their promoters, or both. In vivo bioluminescence imaging (BLI) uses the energydependent emission of visible photons from various luciferase enzymes to detect the location and level of expression of the tagged biological process [5,6]. By contrast, fluorescence imaging uses a fluorescent protein or dye that is excited with external illumination and the excitation is detected externally [4]. The wavelengths of excitation and emission for green fluorescent protein (GFP), and its derivatives, have limited penetration in mammalian tissues owing to absorption, compared with longer wavelengths [7].Therefore, GFP is well suited to high-resolution analyses at the tissue surface – 1–2 mm depths in mouse models [8] – or after replacement of the skin with more transparent materials [9]. Because mammalian tissues absorb light that is used to excite these fluors, the tissues also fluoresce when excited at these wavelengths.The combination of absorption of specific signal and autofluorescence of tissues can result in poor signal-to-noise ratios. A variety of imaging modalities is available for in vivo cellular and molecular imaging [4], and here we highlight http://www.trends.com

the advances made in the area of in vivo BLI and discuss the potential of combining this modality with other modalities for in vivo gene expression studies. In vivo BLI In vivo BLI monitors biological processes by labeling with one of many luciferase enzymes (derived from bacteria, marine crustaceans, fish or insects) that can generate visible light through the oxidation of an enzyme-specific substrate in the presence of oxygen, energy (usually ATP) and cofactors like magnesium [10]. The range of spectral peaks for luciferases characterized to date is 460–630 nm. Among them, the luciferase from the North American firefly (luc) has been most thoroughly characterized; it emits light with a significant red component that can be used for in vivo imaging.The enzyme has been well adapted for use in mammalian cells, for example, as an indicator of cell proliferation, gene delivery or gene expression in cell culture. In addition, it has been used in living animals as a transgenic marker or as a tag for cells that have been transplanted [2].

Caitlin E. O’ConnellRodwell, Stacy M. Burns and Michael H. Bachmann Division of Neonatal and Developmental Medicine,

Background Bioluminescence imaging developed as a result of the synthesis of two technologies – namely, luciferases as biological reporters [10] and ultra-sensitive charged coupled device (CCD) cameras for low-light imaging [11]. The feasibility of using this approach was first shown in a bacterial infection model, where Salmonella were labeled with a set of genes (lux operon) that encode the heterodimeric bacterial luciferase (Lux) and enzymes that can synthesize the substrate for this enzyme. Expression of the five lux genes resulted in light emission without the addition of exogenous substrate, and appeared not to affect bacterial pathogenicity [5]. Because the genes are replicated with each cell division, the entire course of infection could be

0167-7799/02/$ – see front matter ©2002 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(02)02001-2

Dept of Pediatrics

Christopher H. Contag* Division of Neonatal and Developmental Medicine, Dept of Pediatrics, and Depts of Microbiology and Immunology, and Radiology, Stanford University Medical Center, Stanford University, Stanford, CA 94305-5208, USA. *e-mail: ccontag@cmgm. stanford.edu

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Figure 1. Spatio-temporal analysis of transgene expression in living mice (a) Reaction of heme oxygenase-1 (HO-1)-luc transgenic mouse to treatments of CdCl2. Increased HO-1 transcription occurred in the left thigh muscle of 8-week-old HO-1luc Tg mice following treatment with 20 µl of 2 mM CdCl2. Mice were imaged at 0, 3, 6, 9, 12 and 24 h (0, 9 and 24 h time points shown). (b) Western blot analysis of thigh muscle. Thigh muscle was excised and homogenized at each time point and 50 µg of total protein from each sample from the 24 h time point was analyzed. Some variation in expression occurs within the homozygous line including expression in the lymph nodes [as seen in one of the mice in the second panel of (a)]. Figure reproduced, with permission, from [14].

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followed. In this and subsequent studies, the intensity of the emitted light correlated well with bacterial concentration in tissues, with the exception of those tissue sites where oxygen, which is required for light emission, was limited.A wide variety of bacterial lines and DNA constructs are now available for use with BLI in studying bacterial pathogenesis in vivo [12,13]. To monitor the expression of mammalian genes, multiple transgenic mouse lines have been created in which the transgene consists of a regulated promoter fused to the coding sequence of a modified firefly luciferase.The optical signature of the transgene can be detected in living animals and it enables rapid identification of transgenic founder mice [14]. Transgene expression can be studied over time in mice of all ages, and in response to various stimuli. In one model, a transgenic mouse containing the heme oxygenase-1 (HO-1) promoter driving luciferase expression was used to monitor increased HO-1 transcription in response to heavy metal stress (treatment with CdCl2; Fig. 1), and to other treatments [15]. BLI in DNA-based therapy Bioluminescence imaging enables the continuous, real-time monitoring of signals in strategies that use nucleic acids as therapies in vivo. The signals show that optimal delivery and dose has been achieved, and indicate the level of expression of transferred genes [16]. Furthermore, these data are obtained with spatial information such that the sites of effective delivery can be readily localized. For example, a recombinant adeno-associated viral vector expressing luciferase was used successfully to determine the efficacy, safety and long-term expression of foreign genes delivered to fetuses of mice in utero [17].The detection of light from the luciferase reaction in fetal animals in utero indicated that the substrate, luciferin, crosses the placental barrier, and detection throughout the first 18 months of age suggested

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that this delivery method resulted in life-long expression of the reporter gene [17]. Similarly, gene transfer to skeletal muscle [18] and tumor sites [19] has been evaluated. Delivery of foreign genes to liver cells via intravenous injection of DNA [20,21] has been evaluated by using BLI. These gene-delivery studies revealed long-term expression of the transferred gene(s), and indicated that the form of DNA delivered via tail-vein injection does not appear to significantly affect long-term expression levels. Mice that received plasmid DNA in supercoiled or linearized form, or the gene cassette alone, after the plasmid backbone was removed, showed no difference in expression levels over a period of 190 days, the only exception being at the initial 2 h time point (Fig. 2a). These data are in contrast to findings that show a 10–100-fold increase in protein expression from the linear form of DNA versus closed circular DNA [22]. Long-term expression following transfection of the transgene is shown in an image taken at two years post-transfection (Fig. 2b). BLI and cell trafficking Combining cell-based and molecular therapies is a powerful approach for tissue-specific delivery, and understanding cell-trafficking patterns is essential for developing these new approaches. BLI has been used to monitor trafficking patterns of immune cells, and information from these studies has been used to optimize the local delivery of immunoregulatory proteins to sites of autoimmune reactivity. For example, Nakajima and colleagues [23] transduced collagen-specific T cells with a vector encoding a dual-function reporter gene and an interleukin 12 (IL-12) antagonist, IL-12 p40.When co-labeled with GFP–luciferase reporter gene, the collagen-specific T cells could be sorted using a fluorescence-activated cell sorter. These cells were then shown by BLI and reverse transcription–polymerase chain reaction (RT–PCR) to localize to inflamed joints in http://www.trends.com

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mice that had been immunized with collagen peptides.The clinical score for joint stiffness was significantly reduced in mice that were given T cells expressing the immune modulatory protein. Similar results were obtained for myelin basic protein-specific T-cell clones used in experimental allergic encephalomyelitis as an animal model for multiple sclerosis in humans [24]. Multiple BLI reporters The use of multiple reporters in vivo and the opportunity for multiplexing in vivo assays such that two or more parameters can be analyzed in a given animal subject is appealing. Progress was recently made in this area using luc from the firefly and from the sea pansy Renilla reniformis, a blue-emitting luciferase that uses the substrate coelenterazine. Cells labeled with either R. reniformis or firefly luc were injected into mice as a demonstration of tracking two different molecular events in a single animal [25]. Although the potential for this technique is exciting, the pharmacokinetics of the substrate for R. reniformis luc, coelenterazine, appears to be limiting in vivo because it might degrade or be inactivated quickly.The differences between the two available substrates, coelenterazine and luciferin, might make high-throughput studies difficult at this time. However, the short half-life of coelenterazine, and perhaps one of its derivatives, might enable sequential imaging of the two luciferases and enable imaging of two biological processes using BLI. The optical properties of mammalian tissue, which absorbs and scatters most of the light below 600 nm, make distinguishing the two different emission spectra for these enzymes very difficult, hence the ability to separate them biochemically using the two different substrates enables this type of study. http://www.trends.com

Advantages of BLI In vivo biological assays that use luciferase detection are sensitive and rapid. As few as 1000 luciferase-expressing tumor cells, broadly distributed in the peritoneal cavity or circulating after intravenous injection, can be detected using BLI [26]. In addition, luciferase has a high turnover rate, enabling real-time measurements of biological processes to be made [18]. Linear relationships between enzyme levels, as measured by signal intensity and biochemical assays, and tumor load or cell number have been noted in several studies [18,19]. These relationships have enabled rapid determination of log kill of tumor cells after chemotherapy [19], efficacy of immune cell therapies for neoplastic disease [27] and efficiency of gene delivery in vivo. BLI is a broadly applicable technology for assessing biological processes in vivo. Considerations in using BLI The limitations of all optical imaging modalities are twofold, scattering and absorption. Scattering of light at multiple interfaces such as cell and organelle membranes results in highly diffuse emitted light and low spatial resolution. Absorption of light, primarily by hemoglobin, reduces the signal intensity. The transmission of light through scattering and absorbing media has been intensively studied, and the data from these studies can be used to predict the extent to which light, at a given wavelength, is transmitted through mammalian tissues [28].The limited transmission of light through tissue and the requirement of BLI for expressing foreign proteins in the target tissue present some hurdles for transferring this technology to human studies. By contrast, significant advancements in

Figure 2. Comparison of supercoiled, linear and fragment DNA in in vivo transfection experiments Supercoiled or linear plasmid DNA, or the promoter-reporter fragment DNA of pCMV-Luc (in the presence of carrier DNA) were injected intravenously at equal molar concentrations into three groups of four Balb/c mice. Method adapted from Refs [2] and [21]. (a) Anesthetized mice were administered luciferin (150 mg kg−1) and expression levels assessed via bioluminescence imaging and then plotted. A stable level of reporter gene expression was observed after 15 days. The different forms of DNA did not differ in their expression levels except at the 2 h time point. (b) Two mice that received linear DNA were imaged at two years post-transfection, showing the long-term expression of the transgene.

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the chemistry and strategies using clinical imaging modalities have been made that will enable molecular and cellular analysis in humans [4]. An advantage of radionuclide-based methods is the ability to image low levels of reporter gene expression from weak promoters with great precision, such that rate constants of underlying biochemical processes can be quantified [3]. In addition, tomographic information can be obtained with high sensitivity [29]. Although PET offers greater spatial resolution, higher sensitivity and is easier to quantify than SPECT, required radiopharmaceuticals and imaging systems for SPECT are more readily available [4]. Human studies will keep radionuclide-based imaging strategies at the forefront of imaging gene expression [4]. Bioluminescent reporter genes offer several advantages over radionuclide-based reporter genes for real-time analysis of gene expression in animal models, such as inexpensive fine temporal analyses, rapid data acquisition – from seconds to several minutes – labeling versatility, accessible instrumentation and a high signal-to-noise ratio [26]. In addition, the labeling versatility enables a wide variety of biological processes to be studied without the need to develop a new reporter probe for each event. MRI images currently require large amounts of magnetic label and, owing to the time required to obtain high resolution, are best suited to imaging specific regions of interest, such as tumors [4]. In addition, although multiple reporter genes can be used with different modalities such as PET and MRI, they tend to be more labor intensive and time comsuming and might not be well suited to highthroughput studies [25]. In comparison to more penetrating radiation, however, calculating the depth of origin of an optical signal is more difficult. Nonetheless, 3D localization of optical signals has been shown and, with further development, 3D reconstructions from BLI might be possible. BLI and other optical imaging strategies are convenient and are easily adapted to high-throughput animal studies [25], making these approaches attractive for large drug-screening and drug-development programs. Future directions in in vivo gene expression imaging Some researchers have begun to merge different imaging modalities to maximize the strengths of relevant technologies. Recent studies have combined functional imaging (PET) with high-resolution localization (MRI) [30], as well as combining each of these techniques with the high sensitivity of BLI [19,29]. MRI has also been combined with fluorescence imaging [31]. Combining spectroscopic analyses and imaging might also be an exciting area of development in in vivo imaging of gene expression.

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Multiphoton laser scanning microscopy (MPLSM) has been used to improve imaging depth and 3D resolution, and to relieve much of the phototoxicity problems that can be associated with fluorescence microscopic imaging in skin-fold chambers [32]. MPLSM has been used successfully to image cells labeled with GFP at 50 µm depths into a tumor using an intravital window [9]. A miniaturized microPET system has been developed for imaging small animals [33] using a reconstruction image resolution of 1.8 mm in all three axes, which is fully quantitative [34]. Developing areas of research in PET technology include the use of oligonucleotides with mechanisms for signal amplification. These would bind mRNA transcripts directly to detect levels of expression [35].The use of radiolabeled antibodies that target specific proteins that are expressed on the cell surface [36] could be another promising approach. Further advances in fluorescence techniques [1], and confocal [37], multiphoton [32], solid emersion [38] and sub-wavelength resolution near-field [39] microscopy will probably lead to additional breakthroughs in in vivo imaging technology. These will serve as excellent partners in combined imaging strategies because they provide highresolution functional and structural information and, when guided by whole-body scans, can be focused on specific target tissues and cells. Concluding remarks Combined imaging modalities that build on the strengths of each modality are being developed. Combinations that produce high-resolution structural images (MRI) and good functional data (PET, fluorescence or BLI) will be essential as the field of molecular imaging moves forward [19]. In addition, technologies such as quantum dots and others that were previously developed for industrial manufacturing, hold promise for biological applications [40]. Further innovations are likely to come from merging high-resolution microscopy, such as near-field microscopy, with spectroscopic techniques such as Raman spectroscopy [39] and Fourier transform infrared (FTIR) spectroscopy [41]. The nascent field of in vivo cellular and molecular imaging is an exciting area of development, and optical imaging strategies will be at the core of this emerging field. Acknowledgements This work was funded, in part, by grants from the National Institutes of Health (grant numbers 2RO1 HL58013; 1 P20 CA86312-01; 1 R33 CA88303) and the US Airforce Office of Scientific Research (contract number F4962000-1-0349), as well as unrestricted gifts from the Mary L. Johnson and Hess Research funds. http://www.trends.com

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