Noninvasive Bioluminescence Imaging in Small Animals

Abstract

There has been a rapid growth of bioluminescence imaging applications in small animal models in recent years, propelled by the availability of instruments, analysis software, reagents, and creative approaches to apply the technology in molecular imaging. Advantages include the sensitivity of the technique as well as its efficiency, relatively low cost, and versatility. Bioluminescence imaging is accomplished by sensitive detection of light emitted following chemical reaction of the luciferase enzyme with its substrate. Most imaging systems provide 2-dimensional (2D) information in rodents, showing the locations and intensity of light emitted from the animal in pseudo-color scaling. A 3-dimensional (3D) capability for bioluminescence imaging is now available, but is more expensive and less efficient; other disadvantages include the requirement for genetically encoded luciferase, the injection of the substrate to enable light emission, and the dependence of light signal on tissue depth. All of these problems make it unlikely that the method will be extended to human studies. However, in small animal models, bioluminescence imaging is now routinely applied to serially detect the location and burden of xenografted tumors, or identify and measure the number of immune or stem cells after an adoptive transfer. Bioluminescence imaging also makes it possible to track the relative amounts and locations of bacteria, viruses, and other pathogens over time. Specialized applications of bioluminescence also follow tissue-specific luciferase expression in transgenic mice, and monitor biological processes such as signaling or protein interactions in real time. In summary, bioluminescence imaging has become an important component of biomedical research that will continue in the future.

Introduction

The imaging environment for biomedical research has improved significantly in recent years. It is now common for major universities and biomedical research companies to provide researchers with access to a variety of small animal imaging instrumentation. These include x-ray computed tomography (CT), magnetic resonance imaging (MRI¹), magnetic resonance spectroscopy (MRS), positron emission tomography (PET,¹ or microPET), single photon emission computed tomography (SPECT,¹ or microSPECT), ultrasonography, and optical imaging, including fluorescence and bioluminescence. Although the anatomy and function of organs remain important in the evaluation of animal models, molecular imaging is emerging as an increasingly useful component to aid in the understanding of basic biological processes and disease pathogenesis and to monitor therapeutic interventions.

Molecular imaging cannot be precisely defined in a way that would be agreed upon by all imaging scientists, but there would be general agreement that it includes the use of reporter constructs. These are the genetically encoded instructions to manufacture a protein, which is an integral part of the mechanism for imaging contrast. Bioluminescence (also called chemiluminescence) imaging requires a reporter construct to effect production of a protein called luciferase, an enzyme that provides imaging contrast by the light emission that results from the luciferase-catalyzed conversion of D-luciferin to oxyluciferin. The luciferase is called the reporter because it “reports” its location by emission of light under the appropriate conditions. The purpose of this article is to provide information about the basics of bioluminescence imaging, to describe examples of routine and specialized research applications, and to discuss advantages and disadvantages, with an emphasis on the imaging of molecular processes.

The Basics of Bioluminescence Imaging

As mentioned above, bioluminescence imaging in animal models requires a reporter construct that leads to production of the luciferase enzyme. The most commonly used reporter for this purpose is a construct that can express firefly luciferase, normally a heat-unstable enzyme with a biological half-life of approximately 2 hours that must be continuously produced in order for imaging to be accomplished. There are heat-stable variants of firefly luciferase that have special applications due to their greater light emission, but they are not used routinely (Law et al. 2006).

The chemical reaction of the bioluminescence process that leads to light emission also requires magnesium (Mg) and adenosine triphosphate (ATP) in addition to the substrate D-luciferin. Typically the animal receives the D-luciferin in an intravenous (i.v.) or intraperitoneal (i.p.) injection at saturating levels of the luciferase reaction, for example in doses of about 2.5 mg for a 25-gram mouse (100 mg/kg body weight). Alternate methods of delivery include an osmotic pump (Gross et al. 2007) or the addition of D-luciferin to the animal’s drinking water (Gross et al. 2007; Hiler et al. 2006). Imaging of the whole animal can be conducted 10 to 15 minutes after an i.p. dosing of D-luciferin, with relatively stable light emission levels for 30 to 60 minutes, depending of course on the experimental conditions. D-luciferin readily crosses the blood-brain barrier and other body compartments, although injection into the lumen of the gut (a bad i.p. injection) prevents ready absorption.

There are other luciferases besides the firefly variety, including Renilla luciferase (Bhaumik and Gambhir 2002) and luciferase from the nematode (Burns-Guydish et al. 2005; Frackman et al. 1990; Siragusa et al. 1999). Each luciferase has its own substrate specificity, characteristic wavelength of light emission, and optimal parameters (Zhao et al. 2005). More than one luciferase can be imaged simultaneously; for example, Renilla luciferase and firefly luciferase can be used together with minimal interference. There are several Renilla luciferase substrates (coelenterazine analogs) that differ in their properties of autoluminescence. The route of delivery in the animal is also important for coelenterazine derivates; intravenous delivery produces the highest peak signal but with the shortest duration (Zhao et al. 2004). With the nematode luciferase applied for imaging bacteria, the complete set of enzymes for production of the substrate is also encoded so that the injection of a substrate is not necessary.

For all luciferase reporters, the goal is to detect the light emitted from the animal. This is accomplished by placing the animal in a dark chamber and using a charge-coupled device (CCD¹), which is a light-sensitive camera. The IVIS-100 system and Living Image Software (both from Xenogen, Inc., now called Caliper LifeSciences) are commercially available for this purpose. Mice are normally under anesthesia during imaging to prevent movement, with heat provided to maintain body temperature.

The time to acquire images ranges from 1 second to 10 minutes, depending on the experiment. Data acquisition software ensures that no pixels are saturated during image collection. The intensity of light emission is represented with a pseudo-color scaling of the bioluminescent images. These false scales assign colors for the different rate of light emission; for example, the red areas show the highest amount of light emission, and blue/violet areas show the least. The bioluminescent images are typically overlaid on black and white photographs of the mice that are collected at the same time. In this manner it is easy to determine the precise location of the light emission. Region-of-interest software (provided by the vendor) measures the amount of light emission from a given area (relative or absolute). When relative counts or photons are used they are typically normalized to acquisition time and presented as counts (or photons) per second (or minute).

It is important that acquisition and analysis parameters (e.g., animal position relative to CCD camera, f-stop, region of interest) remain constant when relative counts or photons are used. The absolute unit of radiance is photons/sec/cm²/steradian, and refers to the photons per second of light that radiate from the mouse in a unit area (1 cm²) and unit angle (1 steradian). This more quantitative unit enables easier comparison between systems and experiments, and allows the comparison of data in an experiment when image acquisition parameters are greatly different.

Routine Research Applications

It is convenient to divide the research applications of bioluminescence into routine and specialized. For routine applications, it is typical for the luciferase reporter construct to include a control element (promoter) that is always active, or “on” under all conditions, in all tissue types. One example is the cytomegalovirus (CMV¹) immediate-early promoter, which is highly active in most tissues. A summary of research applications is provided in Table 1.

Table 1.

Summary of major bioluminescence applications in small animals

Application type	Indication	Purpose
Routine	Cancer	Detection of tumor location, monitoring growth
	Gene therapy	Targeting of vectors
	Cell therapy	Migration of adoptively transferred cells
	Infection	Clearance of pathogens (bacteria, virus, etc.)
Specialized	Tissue-specific promoters	More specific control of reporter expression that is responsive under defined conditions
	Protein-protein interactions	The study of biological pathways
	Bioluminescence resonance energy transfer (BRET)	The study of biological pathways
	Apoptosis detection	Evaluation of new cancer therapies
	Viral replication	Oncolytic cancer therapy
	Signal transduction	Real-time monitoring of biological pathways, as influenced by various treatments
	Transgenic mice	Understanding of gene regulation, or the study of other biological processes

Plasmid or viral techniques are effective for the transfection of cancer cells with the luciferase reporter construct. Figure 1 shows an image of a firefly luciferase–positive xenografted tumor in the mammary fat pad of a mouse. The human breast cancer cells (2LMP, a subclone of MDA-MB-231) were stably expressing the luciferase gene under control of the CMV promoter, using an AAV vector. The cells were detected in the mammary fat pads of nude mice and grew into tumors whose size was reliably assessed noninvasively by serial bioluminescence imaging. Using this approach, there was excellent correlation between tumor size and light emission (Figure 1). This method has also been effective in evaluations of novel therapies for ovarian cancer and for animal models of prostate cancer (Brakenhielm et al. 2007; Kanerva et al. 2003, 2005).

Figure 1.

(A) Bioluminescence imaging of a representative mouse at 7 days after implantation of luciferase-positive 2LMP cells in the mammary fat pad. (B) The summary graph (based on imaging and measurements of 5 mice) comparing bioluminescence with tumor measurements of volume using calipers. Imaging data and caliper measurements were normalized to the initial values for each mouse and hence begin at 100%. The original figure is in color and is available in the online posting of this article at www.ilarjournal.com.

Bioluminescence imaging also has sufficient sensitivity to detect luciferase-positive tumor cells immediately after implantation, weeks before the tumors could be visually detected for caliper measurements. As shown in Figure 1, the tumors could be measured after 19 days, but bioluminescence imaging detected the 1 million tumor cells immediately after implantation, when the level of light emission averaged 11,600 relative photon counts per second in the tumor region of interest, and background counts were less than 1% of that level. The animal received luciferin (2.5 mg/mouse, i.p.) 15 minutes before imaging. In terms of detection limits for cell numbers, it was relatively easy, with this particular breast cancer line, to detect 5,000 cells implanted subcutaneously.

Naturally, the detection limit would also depend on the location of the cells and the level of basal luciferase expressed in each transformed cell. For intracardiac injection (left ventricle), 200,000 cells were easily detected with bioluminescence images collected for 3 minutes, as shown in Figure 2A.

Figure 2.

Bioluminescence imaging of 200,000 luciferase-positive MDA-MB-435 cells immediately (A,C) or 33 days (B,D) after injection into the left (A,C) or right (C,D) ventricle. Disseminated metastases or focal lung lesions are shown resulting from left or right ventricular injection, respectively. Imaging times vary due to saturation at later acquisition time. A 120-second acquisition was taken for both day 0 images (A,C); day 40 images required only a 10-second duration (B,D). The original figure is in color and is available in the online posting of this article at www.ilarjournal.com.

Visual inspection permits the eventual detection of subcutaneous tumors, but it is not possible to easily detect metastasis of cancer cells. Bioluminescence imaging is particularly sensitive for this purpose, as shown in Figure 2. MDA-MB-435 breast cancer cells were induced to express firefly luciferase using a lentiviral vector (as described in Cowey et al. 2007) and were administered by intracardiac injection. The image in Figure 2A was collected immediately after injection into the left ventricle and shows the cells in the blood pool. The image in Figure 2B was collected approximately 42 days later, and shows the location of metastatic disease in the spine, joints, and head (mandibles). Figure 2C shows an intracardiac injection in the right ventricle, with cells localizing to the lung. In this particular model the cells did not distribute to other locations (Figure 2D). Tumor burden during treatment was monitored weekly during this study.

If viral vectors encode the luciferase reporter, bioluminescence imaging can detect the cells that become infected after dosing. Viral vectors of many types are effective for gene therapy, and are constantly being improved to be more specific for the targeting of certain tissues or human cancers. One widely used vector is the serotype 5 human adenovirus (Ad5¹), rendered replication incompetent by removing parts of the viral genome, thereby allowing space in the viral genome to insert genes for imaging or therapy. Figure 3 shows a cartoon of three different routes for the administration of an Ad5 vector encoding firefly luciferase. Infection of the cell is via the coxsackie and adenovirus receptor (CAR) and leads to production of luciferase enzyme in the cytosol and subsequent light emission when luciferin is provided.

Figure 3.

A diagram showing injection of the replication-incompetent Ad5 vector by three different routes, together with a figure showing the mechanism of luciferase expression in an individual cell. CAR, coxsackie and adenovirus receptor; CMV, cytomegalovirus promoter; luc, luciferase.

As shown in Figure 4A, imaging at 6 hours after dosing with Ad5 encoding firefly luciferase (1 × 10⁹ plaque forming units, pfu) detected the expression of luciferase in the liver for i.v.-dosed nude mice, or in the peritoneal cavity for i.p.-dosed nude mice. The liver expression persisted for a longer time, as shown with an image collected 13 days after dosing (Figure 4B), whereas luciferase expression in the i.p.-dosed mice was not detected at this time. A summary of the mean relative counts per second (with error bars as standard deviation) for the two routes of administration is summarized in Figure 4C. This type of long-term imaging is applicable in immune-competent mice as well, and was an important tool to establish the importance of complement in liver transfection after intravenous delivery in B57BL6 mice (Zinn et al. 2004).

Figure 4.

Bioluminescence imaging at (A) 6 hours or (B) 13 days after dosing with the replication-incompetent Ad5 vector encoding luciferase, together with (C) a summary graph of mean light emission (error bars are standard deviations) for all mice at all imaging time points. i.v., intravenous; i.p., intraperitoneal. The original figure is in color and is available in the online posting of this article at www.ilarjournal.com.

Other routes of administration are also amenable to imaging, as shown in Figure 5A for mice dosed intratracheally. After intubation, normal B57BL6 mice were dosed with 4 × 10⁸ pfu of the Ad5, encoding both firefly luciferase and the human type 2 somatostatin receptor (hSSTr2), each under control of a separate CMV promoter element. Luciferase expression in lungs as detected by bioluminescence imaging showed infection in the lungs of the three mice, and this was confirmed by SPECT/CT imaging of an hSSTr2-avid, Tc-99m-labeled peptide, as shown in Figure 5B. A control mouse without Ad5 dosing did not show uptake of the same radiolabeled peptide (Figure 5C). (The method for imaging the hSSTr2 reporter has been reported in Buchsbaum et al. 2004, 2005, and Zinn et al. 2002.) An alternate approach to this example of using two reporter constructs controlled by separate promoters for multimodality imaging is to fuse the luciferase to imaging reporters that are specific for other imaging modalities such as fluorescence or PET (Nishii et al. 2006; Ray et al. 2007).

Figure 5.

Imaging of (A) luciferase expression at 24 hours after controlled intratracheal delivery of replication-incompetent Ad-Luc-hSSTr2 (4 × 10⁸ pfu) in three BL/6 mice, (B) mouse #3 by SPECT/CT 5 hours after i.v. hSSTr2-avid Tc-99m-P2045 showing Ad-specific hSSTr2 lung expression, and (C) a control mouse without lung hSSTr2 expression. The original figure is in color and is available in the online posting of this article at www.ilarjournal.com.

While the images presented thus far have concentrated on bioluminescence imaging of Ad vectors that induce cells to produce luciferase after infection, a novel fusion of luciferase with a capsid protein also enabled imaging of the actual Ad particle itself (Matthews et al. 2006). This latter approach may have utility for other vectors, and certainly the former approach has been used to detect viruses including adeno-associated virus (AAV), herpes virus, and other pathogens (Burgos et al. 2006; Franke-Fayard et al. 2006; Luker et al. 2003).

Bioluminescence imaging is also applicable to the detection of bacteria (Burns-Guydish et al. 2005; Siragusa et al. 1999). As shown in Figure 6, the colonization of Citrobacter rodentium in wild-type C57BL/6 mice was imaged over 15 days. Figure 6A shows 60-second exposures of a representative member of a group of 10 wild-type C57BL/6 mice. These images and the accompanying graph showing the group means illustrate that bacterial colonization peaked around 5 days after inoculation (PI). Clearance of the pathogen began at day 9 or 10 PI and was complete by days 15 to 17.

Figure 6.

Bioluminescence imaging of Citrobacter, with (A) a representative mouse imaged over time, (B) summary data for all mice in the group (means with error bars as standard deviations), (C) image showing the location of Citrobacter at termination, and (D) image of the containment chamber for imaging. The original figure is in color and is available in the online posting of this article at www.ilarjournal.com.

Citrobacter rodentium, which induces in rodents a transient colonic hyperplasia and diarrhea over the course of 7 to 28 days, has been used as a model of enteropathogenic Escherichia coli virulence infections in humans. Although in immunocompetent mice the disease usually resolves within 30 to 40 days, in neonates and lymphocyte-deficient strains the bacterium often causes significant mortality. C. rodentium attachment occurs through a type III secretion system and results in enterocyte effacement, the earliest sign of infection. Epithelial proliferation, increased colonic crypt depth, and a mixed infiltrate consisting of neutrophils, macrophages, and lymphocytes are characteristic of the subsequent inflammatory response. Finally, bacterial clearance and lesion resolution occur after the induction of a systemic CD4⁺ T cell–dependent immunoglobulin G (IgG) response.

The studies shown in Figure 6 used a bioluminescent strain, C. rodentium ICC180 (generously given by Drs. Gad Frankel and Siouxsie Wiles of Imperial College London, UK), which expresses the luxCDABE operon from the entomopathogenic nematode symbiont Photorhabdus luminescens. Insertion of the luxCDABE operon into the host chromosome was achieved via transposon mutagenesis with a mini-Tn5 vector (Wiles et al. 2004, 2006). As shown in Figure 6C, removal of the abdominal wall and exposure of the cecum and colon revealed that C. rodentium preferentially colonized the cecal lymphoid patch and the mid and distal colon (arrows). For imaging, the mice were placed in a supine position in a custom-built hermetically sealed chamber with a viewing window (Figure 6D). The sealed chamber allowed for containment of the pathogen and thus minimized exposure of other animals being imaged in the facility. The mice were loaded into the chamber in a biosafety cabinet located in the same room as the imaging system.

Specialized Research Applications

Because bioluminescence detects the expression of a genetic reporter (luciferase) there are opportunities to control that expression based on the activity of the promoter element. For example, the liver expression of luciferase in Figure 4A resulted because the CMV promoter element is “on” in hepatocytes. An alternate promoter such as cyclooxygenase 2L (cox-2L) is “off” under normal conditions in the liver, but is sensitive to being turned “on” in the presence of inflammation (Ishikawa et al. 2006). The images in Figure 7 show this approach of turning luciferase from “off” to “on.” Five mice received injections of an Ad vector (1 × 10⁹ pfu) encoding firefly luciferase under control of cox-2L (Wesseling et al. 2001). As shown in Figure 7 for a representative mouse, no liver expression was detected initially, but an injection of lipopolysaccharide (LPS-2 µg) transiently induced the expression of luciferase in the liver. Figure 7 represents a summary of the kinetics of this induction for the entire group over 49 hours. In the case of weak, tissue-specific promoters, others have reported ways to amplify the signal (Iyer et al. 2005, 2006; Ray et al. 2004).

Figure 7.

Imaging of liver expression of luciferase under control of the cyclooxygenase 2L promoter, (A–E) time course before and after lipopolysaccharide (LPS) injection for a representative mouse, and (F) summary graph for all mice in the group. The original figure is in color and is available in the online posting of this article at www.ilarjournal.com.

In addition to control of luciferase expression by specific promoter elements, Laxman and colleagues (2002) described an approach to produce an “inactive” luciferase enzyme by linking a cleavable protein subunit to the luciferase. Within the subunit were specific sequences cleaved by activated caspase 3, thereby making the luciferase molecules “active” only under conditions of apoptosis induction. This reporter was then inserted in cancer cells and used as an early detector of induced apoptosis. This group has reported additional bioluminescence methods to detect apoptosis (Lee et al. 2007; Rehemtulla et al. 2004). Liu and colleagues (2005) have developed a luciferin substrate that was cleaved to an active form after induction of apoptosis. Luker and colleagues (2004) reported another mechanism to convert inactive luciferase to an active form, extending a strategy originally described by Paulmurugan and colleagues (2002), to produce separate “inactive” portions of the luciferase enzyme that combined either spontaneously or in response to a drug to form an “active” holoenzyme only under conditions of specific protein-protein interaction. The complementation imaging approach was applied to follow protein-protein interactions in cells and live animals. The system has been improved to now include self-complementing luciferase constructs (Paulmurugan and Gambhir 2005). Finally, future methods may exploit the process of bioluminescence resonance energy transfer and enable luciferase to drive fluorescent light emission (Nishii et al. 2006; So et al. 2006).

The increase in bioluminescence signal after the spread of a replication-competent vector has allowed detection of breast xenografts. Mice with subcutaneous (s.c.) xenografted breast tumors were imaged after i.v. injection of a replication-competent Ad vector encoding firefly luciferase (Ad5-Luc). As expected, expression of luciferase was detected in the liver within 24 hours of the injection. Luciferase expression in the liver decreased over time, but increased in the s.c. tumor site due to replication of the Ad vector at that location. The images in Figure 8 show luciferase expression increasing in the tumor between 2 and 3 weeks after intravenous injection of the replication-competent vector. Others have reported detection of xenografted tumors with the i.v. delivery of targeted, replication-incompetent Ad vectors (Niu et al. 2007).

Figure 8.

Imaging luciferase expression in a breast xenograft at (A) 2 weeks and (B) 3 weeks after intravenous injection of a replication-competent Ad5 vector encoding luciferase. The luciferase expression in the tumor was visualized due to replication of the vector. The pseudo-color scale bars at the bottom represent the intensity of light emission with different colors. The original figure is in color and is available in the online posting of this article at www.ilarjournal.com.

Hepatic growth hormone (GH¹) signaling served as the model system for development of a noninvasive system to study in vivo signaling (Frank et al. 2006). GH is a prime regulator of postnatal growth and metabolism in vertebrates. It affects numerous tissues, but has major effects in the liver. The developed system uses a signal transducer and activator of transcription-5 (STAT5)-dependent luciferase reporter (a GH response element, GHRE), introduced into a nude mouse liver by adenoviral injection. A cartoon outlining the system is shown in Figure 9.

Figure 9.

Diagram showing mechanism of growth hormone–induced expression of luciferase. CAR, coxsackie and adenovirus receptor; GHR, growth hormone receptor; GHRE, growth hormone response element; JAK2, Janus kinase 2; luc, firefly luciferase; STAT5, signal transducer and activator of transcription-5; Yp, phosphorylated tyrosine residue; TTC-NNN-GAA, DNA response element for STAT5.

Noninvasive bioluminescence imaging was used to detect GH-induced STAT5-dependent hepatic GH signaling serially in individual mice, as shown with two different, one-time doses of i.v. GH (Figure 10). In both groups of mice there was significant, time-dependent GH response that peaked 3 hours after GH injection, and the level of the response was dependent on the amount of GH injected (Figure 11). These results indicated the bolus of GH-induced luciferase expression by turning on the GHRE, but only transiently. Because induced luciferase is heat unstable, the light emission from luciferase decreased over time in the absence of the GH. However, as shown in Figure 11, the experiment could be repeated with similar results on separate days. Each day the mice were injected again with GH to induce transient expression of the luciferase. This imaging system allows detailed in vivo analysis of GH signaling and action and serves as a paradigm for studies of additional signaling pathways in liver and other tissues, including tumors. It is also possible to introduce another GH receptor, normal or modified, and thereby study signaling from the introduced GH receptor to the GH response element that drives luciferase (Frank et al. 2006).

Figure 10.

Imaging of growth hormone (GH) signaling over time. Two representative mice from each dosing group (n = 10/group) are presented. The original figure is in color and is available in the online posting of this article at www.ilarjournal.com.

Figure 11.

Liver-induced luciferase signal in the two groups of mice dosed with growth hormone (GH) (from Figure 10). Data are expressed as means with error bars as standard deviations. The mice were injected with GH on 4 different days and imaged.

A new transgenic mouse model provides a tool in the tracking of naïve T-cell populations in real time (Azadniv et al., in press; Dugger et al. 2004). This model entails the cloning of the firefly luciferase gene into human cd2 genes, limiting constitutive expression to all T cells. Luciferase imaging of sorted cell populations provides evidence for the T-cell-specific expression of the luciferase gene in both CD4+ and CD8+ T cells. Imaging of an intact cd2.luc.tg mouse permitted delineation of primary and secondary lymphoid organs, as shown in Figure 12A. Imaging easily detected the in vivo location of 3.5 × 10⁶ cd2.luc.tg T cells after adoptive transfer into CD45.1 C57.B6 mice, as illustrated in Figure 12B. This image was collected 3 weeks after injection of the cd2.luc.tg T cells, and individual lymph nodes were clearly visualized. For these experiments it was necessary to feed the mice a casein-based diet (Formula 89222, Harlan Teklad, Madison, WI) to reduce the background signal by a factor of 200, as compared with that from normal mouse chow with plant material that showed greater phosphorescence.

Figure 12.

Bioluminescence imaging of luciferase-positive T cells in (A) a founder mouse and (B) representative mouse at 3 weeks after adoptive transfer of 3.5 × 10⁶ luciferase-positive T cells. The original figure is in color and is available in the online posting of this article at www.ilarjournal.com.

In order to define key regulatory regions of the melanocortin- 4 receptor (MC4R¹) gene, a series of transgenic mice were created with various MC4R promoter fragments that were linked to a luciferase reporter gene and then imaged in the live animal. The activity of “neural” melanocortin receptors (MC3R and MC4R) is tightly regulated by ligands secreted by distinct populations of anorexigenic and orexigenic arcuate nucleus neurons that project throughout the brain and produce either agonists (alpha-MSH and gamma-MSH, derived from the proopiomelanocortin or POMC gene) or an antagonist (agouti-related protein or AGRP), respectively. Preliminary analyses indicate that proximal promoter elements (within 900 bp) are sufficient to drive brain-specific expression of the luciferase transgene.

In general, bioluminescence imaging indicated that several founder animals with 3,300 to 890 bp of promoter sequence display detectable luciferase activity in brain and midline regions (presumably spinal cord) as expected (Figure 13A). Additionally, several founder animals display expression in other tissues, presumably due to positional effects imposed on the promoter constructs when integrating next to strong enhancer elements (e.g., an apparent liver-and kidney-specific enhancer imparts a near “saturation” signal of bioluminescence; Figure 13B). Although only a few founder animals (n = 4) have been created and imaged with the shortest promoter construct (430MC4Luc), only slight activity has been detected above background (and fortunately MC4R has not shown any activity in the dermis covering the head). The representative founder transgenic mice shown in Figure 13 illustrate how bioluminescence imaging can detect tissue-specific gene expression even from the weak MC4R promoter. The ability to assess the activity of the MC4R promoter in vivo enables an understanding of how this important feeding gene is regulated, especially since an appropriate tissue culture model does not exist.

Figure 13.

In vivo imaging to screen MC4R-transgenic mice driving luciferase. Shown are founders from four different promoter constructs. (A) Founder C1 (3300MC4Luc), prominent activity pattern along midline and brain. (B) Founder B5 (3300MC4Luc3), very strong expression in liver and kidneys. (C) Founder D2 (890MC4Luc), prominent activity in forebrain, weaker along midline. (D) Founder E1 (430MC4Luc), weak activity in abdominal area (not shown) and paws. The original figure is in color and is available in the online posting of this article at www.ilarjournal.com.

These two examples of transgenic mice with luciferase constructs represent only a small fraction of those that are commercially available or have been developed in academia (Hsieh et al. 2005; Lyons et al. 2003; Malstrom et al. 2004; Roth et al. 2006; Sadikot et al. 2002, 2003). There are potential difficulties in establishing transgenic mice with luciferase constructs, because positional effects can cause aberrant results, as shown in Figure 13B, especially when using weak promoters. To overcome this potential problem, it is useful to establish and analyze multiple founders that contain the promoter fragment of interest, use transgenic “insulator” elements, or use gene-targeting approaches when feasible.

Sensitivity of Bioluminescence

The parameters that influence sensitivity in bioluminescence imaging are the following:

• instrumentation,

• hair,

• depth in tissue,

• imaging parameters, including acquisition time, binning, and animal position,

• level of promoter activity controlling luciferase expression,

• amount of luciferase substrate injection, and

• diet (gut phosphorescence increases background noise).

It is essential to optimize these parameters for each experiment. Of primary importance is the instrumentation, especially the CCD camera, which is normally cooled to −105°C to reduce background noise. The presence of hair on the animal and the depth of the tissue are also important factors. A position close to the CCD camera, longer acquisition times, and higher binning all increase sensitivity. Depending on the experimental conditions, the promoter activity driving luciferase as well as the amount of luciferin injection can have a significant effect. Reducing the background signal enables the detection of lower levels of light emission from luciferase; for example, as mentioned above, a diet without plant material can reduce background phosphorescence as compared with standard mouse chow. Finally, if anesthesia has decreased the animal’s body temperature, there will be less light emission from the luciferase reaction; it is therefore important to carefully monitor the animal’s temperature and provide heating to maintain normal body temperature.

Conclusions

Bioluminescence imaging is a versatile and cost-effective technology that has high sensitivity and efficiency. Five mice (or three rats) can be imaged at the same time for most experiments, with acquisition times ranging from a few seconds to 10 minutes; in practical terms this means that it is possible to image 150 to 200 mice or more in a single day. A position closer to the camera increases sensitivity, but permits the imaging of only two mice simultaneously, reducing efficiency. Simple region-of-interest analyses can easily reduce the data. With the appropriate conditions, light is detected only from areas that contain luciferase and there is minimal or no signal in areas where only luciferin is present. Since luciferase has a short biological half-life, it must be continuously produced for detection. When luciferase-expressing cells die, the signal is quickly lost. Similarly, changes in luciferase levels can be detected when promoter elements driving luciferase are turned off or on.

The cost of bioluminescence imaging systems is significantly less than other imaging technologies such as SPECT, PET, or MRI, which image one animal at a time and may require 30 to 60 minutes for data acquisition. In practical terms the total number of mice that could be imaged in a single day would be between eight and ten for most experiments. Further, data analyses are complicated and require more time. PET and SPECT imaging require the preparation and injection of radiolabeled molecules. The imaging contrast for these modalities is provided by the radiolabeled molecule and depends on selective retention, or lack thereof, in the tissue of interest. The imaging systems detect the radioactive label in all locations, including that which is metabolized or excreted by normal routes. This “background” signal often requires imaging to be conducted at later time points when it is reduced, and certainly reduces sensitivity to detect the desired process. However, these disadvantages are offset by the resulting 3D images and the ease in translation to human imaging application.

Bioluminescence imaging provides 2D images that are relatively low resolution, although a new 3D imaging instrument is now available (Virostko et al. 2007). Although a major disadvantage is the lack of translation of this technology to human applications, it can be overcome using alternate reporter constructs that are specific for human imaging technologies such as SPECT or PET. Other disadvantages include the requirement for genetically encoded luciferase, the injection of the substrate to enable light emission, and the dependence of light signal on tissue depth. In practical terms, however, these limitations are not significant for small animal imaging, especially when considered in the context of the many advantages of the technique.