Reporter Genes And Reporter Probes

A. Optical Reporter Systems

There are essentially 4 different types of optical reporter genes currently in use for studies in living animals. These genes either encode a) a protein that contains a chromophore (a short, internal peptide segment that contributes to the protein's fluorescent capabilities following obligatory posttranslational modifications), which fluoresces when externally irradiated (fluorescence imaging), b) an enzyme that can convert an ex-ogenously added, optically quiescent substrate into a fluorescent complex, or an enzyme that changes the conformation of a substrate such that it fluoresces a different color (fluorescence imaging), c) luciferases that generate light when presented with the appropriate substrate (bioluminescence imaging), or d) fusion proteins that couple transgene products with

Reporter Cell

Figure 4 Basic Principles of Magnetic Resonance Imaging (MRI) In MRI, subjects are placed in a strong, external, magnetic field, B0, produced by a hollow, cylindrical magnet. The B0 field is nearly uniform and points parallel to the long axis of the magnet. Imaging with MRI is dependent upon atomic nuclei with an odd number of protons, such as hydrogen (JH). Such atoms have their own net magnetic field [a.k.a., magnetic dipole moment (MDM)] and their moments align accordingly when placed in this external magnetic field. Once equilibrium has been achieved between the subject and the magnet, energy can be added to the system in the form of a radiofrequency (RF) pulse. In most cases, this pulse, which generates its own magnetic field, can change the alignment of the hydrogen atoms such that their moments are now perpendicular to B0. Once the RF pulse is ''turned off'', the hydrogen atoms realign or ''relax'' to B0 and give up energy in the form of RF waves during the relaxation period. Receivers located in the magnet capture this RF wave. One of the calculations made from the captured information is the rate at which the hydrogen atoms relax to equilibrium. Image construction and image contrast are possible with MRI because hydrogen atoms associated with macromolecules like fat and proteins have a significantly different relaxation rate than the hydrogen atoms of bulk water. The measurements of relaxation rates can be converted into a value, which translates into image pixel value, with each pixel representing a small, representative, unit volume of the subject (voxel). On a certain MR imaging protocol called a ''T1-weighted'' sequence, a voxel composed mostly of fatty (hydrocarbons) protons will have a high (bright) signal since the rate of relaxation is rapid. In contrast to voxels that contain a large number of water protons, this voxel will have a low (dark) signal on T1-weighted MR imaging since the rate of relaxation is much longer. See color insert for color version of this figure.

fluorescence or bioluminescence optical reporters with a peptide linker (discussed in greater detail in a later section). When compared with other imaging modalities, fluorescence and bioluminescence imaging techniques hold tremendous potential for study of small living animals because of their relative affordability, relative ease of use, high assay sensitivity, and low requirement for specialized support personnel. In contrast to the radionuclide-based techniques described earlier, all optical reporter gene/probe systems are forms of indirect imaging. The measured light emissions generated from these re-

porter systems may or may not correlate to the amount of therapeutic gene product present. Briefly reviewed below, extensive work in the past several years has been dedicated to mutating existing or cloning new fluorescent or bioluminescence genes that are more compatible for use in living subjects.

Mammalian tissues pose a number of obstacles for the propagation of light and, thus, are a challenge for the evaluation of fluorescence or bioluminescence-based reporter genes in living subjects. Fluorescent-based techniques, in particular, face additional challenges since both the light used to excite the reporter probe and the light emitted from the probe are subject to absorption, scatter, and other optical tribulations. More specifically, the excitation light is not only limited by its ability to penetrate nontransparent tissue, but also contributes to background autofluorescence (from hemoglobin and cytochromes) especially when excitation wavelengths are in the blue and green portions of the spectrum, which is generally the case when green fluorescent proteins (GFP) [and its close relatives such as blue fluorescent proteins (BFP)] are used as reporter genes (21). As a result, fluorescence imaging has relatively poor signal-to-noise when compared to bioluminescent techniques.

Light emitted from either fluorescence-or biolumines-cence-based techniques is subject to tissue absorption and scattering. In mammalian tissues, the blameworthy structures responsible for light absorption are predominantly molecules of hemoglobin, which absorb wavelength emissions of 400-600 nm. To a lesser extent, melanin and other pigmented macromolecules also contribute to light absorption, and, thus, experiments using white-furred or hairless subjects are preferred when using optical reporter systems. Light absorption by water molecules is also another important factor, but not until wavelengths approach >900 nm range. Light scatter is another important confounding factor in the detection of low-level photon emissions; the interfaces at the surface membranes of cells and organelles are largely the cause for this occurrence (21).

Yet, despite these impediments, recent advances in the fields of optics and sensor technology make it possible to detect relatively low emission events with great sensitivity and generate remarkable images (35,36). Furthermore, optical reporters are being developed to operate with longer wavelengths; that is, away from the blue-green part of the spectrum and towards the red (a.k.a. ''red-shifted,'' between 600-900 nm) so as to maximize transmission, minimize absorption, and minimize background autofluoresence. Additionally, the fluorescence efficiency of each of the fluorescent proteins is being optimized through site-directed mutagenesis. The intrinsic physical and chemical properties of the mutants are altered. Characteristics such as protein stability, extinction coefficients, fluorescence quantum yield, tendency to dimerize or form multimers, requirement for oxygen, efficiency of fluoro-chrome formation, and susceptibility to photoisomerization and photobleaching can be modulated through mutagenesis, and, thus, the amount and rate of light photons emitted from such structures can be optimized (37).

For example, wild-type Aequorea GFP, a 238 amino acid polypeptide (27-30 kD) specifically isolated from the Pacific jellyfish (Aequoea victoria), is a highly fluorescent molecule with excitation peaks at 395 nm (largest peak) and 475 nm (38). GFP's emission peak is 509 nm, which is in the lower green portion of the visible spectrum. On the skin surface, GFP expression can be easily visualized. However, for optimal visualization of GFP expression in deeper structures, such as the brain and pancreas, skin or skull windows need to be created by surgical incision (39). Additionally, GFP fluorescence is not immediate and only detectable at about 7 h after injection of recombinant adenovirus carrying GFP (vAd-CMV-GFP). The rate-limiting step in GFP ''maturation'' appears to be a necessary oxidation step in chromophore formation (40).

Extensive work has been dedicated to creating GFP mutants, since wild-type GFP possesses a few factors compromising its use in living subjects. The list of available GFP mutants, each having their own idiosyncrasies, is quite lengthy. An excellent review is available (37). One of the more thoroughly studied mutants S65T, also known as enhanced GFP (EGFP), has some impressive advantages. Ser65, one of the amino acids of the chromophore, is replaced by Thr in this mutant. The wild-type 395 nm excitation peak is suppressed, and the 475 nm peak is enhanced 5- to 6-fold in amplitude (6-fold increase in brightness), the peak is shifted to 489-490 nm (40). It is 4-fold faster during the rate-limiting oxidation step, not subject to photoisomerization, and exhibits very slow pho-tobleaching (41). Examples of the use of EGFP in living subjects is shown in Fig. 5.

GFP mutants come in all kinds of colors: blue, cyan, yellow, and green. However, the maximum emission peak attained by the GFPs is 529 nm. Recently cloned red fluorescent protein (dRFP), a 28-kDa protein responsible for red coloration seen in the coral Discosoma, has broken the 529 nm barrier, and excitation and emission maxima are at 558 nm and 583 nm, respectively (42). The relatively high extinction coefficient and fluorescence quantum yield indicate that the brightness of the mature, well-folded protein is comparable to any other fluorescent protein. Furthermore, a commercially available mutant, DsRed, is resistant to photobleaching and has been further red-shifted to 602 nm, which reduces the tissue absorption of the emitted light photon. Unfortunately, there are some significant limitations to the use of dRFP. It is an obligate tetramer and is quite slow in its maturation—it takes days for it to mature from green to red. Mutations attempted thus far have not been able to alleviate these problems (42). Figure 1 contains a fluorescent image example of an RFP-expressing glioma in a mouse model.

Two recently developed ''smart'' fluorescent probes have been developed that are significantly red-shifted—so much that they work in the near-infrared portion of the electromagnetic spectrum (excitation wavelength 673 nm; emissions wavelength of 689 nm), making them ideal fluorescent agents for use in intact organisms. Although they are being exploited to measure endogenous protein levels, they are worth mentioning here since they have the potential to be activated by exoge-

Figure 5 Optical (Fluorescence) Imaging of Transgene Expression The adenoviral (vAd) vector AdCMV5GFPAE1/AE3 [vAd-green fluorescent protein (GFP)] (Courtesy of Quantum, Montreal, Canada) constitutively expresses an enhanced GFP (eGFP), which is driven by a CMV promoter. The vector was delivered to the brain after an upper midline scalp incision and creation of a parietal skull window. Twenty mL containing 8 x 1010 plaque-forming units (pfu)/mL vAd-GFP per mouse was injected into the brain. Twenty-four hours later, fluorescence imaging of the entire animal (lower magnification) was carried out in a light box illuminated by blue light fiber optics, which provided the external excitation wavelength, and was imaged using a cooled color charge-coupled device camera. Emitted fluorescence was collected through a long-pass filter GG475 on a 3-chip thermoelectrically cooled, color CCD camera. Images of 1024 x 724 pixels are captured directly on a personal computer or continuously through video output on a high-resolution video recorder. Images are subsequently processed for contrast and brightness and analyzed with imaging software. Higher magnification images (not shown here) can be accomplished by using a fluorescence stereomicroscope equipped with a 50 W mercury lamp. In this scenario, selective excitation of GFP is produced through a D425/60 band-pass filter and 470 DCXR dichroic mirror. Emitted fluorescence are captured and processed as described above. Images A and B demonstrate GFP transgene expression following adenoviral delivery to the brain. Image C demonstrates Ad-CMV-GFP delivery to the liver via portal vein cannulation. See color insert for color version of this figure. (Images courtesy of Anticancer, Inc., Ref. 18.)

Figure 5 Optical (Fluorescence) Imaging of Transgene Expression The adenoviral (vAd) vector AdCMV5GFPAE1/AE3 [vAd-green fluorescent protein (GFP)] (Courtesy of Quantum, Montreal, Canada) constitutively expresses an enhanced GFP (eGFP), which is driven by a CMV promoter. The vector was delivered to the brain after an upper midline scalp incision and creation of a parietal skull window. Twenty mL containing 8 x 1010 plaque-forming units (pfu)/mL vAd-GFP per mouse was injected into the brain. Twenty-four hours later, fluorescence imaging of the entire animal (lower magnification) was carried out in a light box illuminated by blue light fiber optics, which provided the external excitation wavelength, and was imaged using a cooled color charge-coupled device camera. Emitted fluorescence was collected through a long-pass filter GG475 on a 3-chip thermoelectrically cooled, color CCD camera. Images of 1024 x 724 pixels are captured directly on a personal computer or continuously through video output on a high-resolution video recorder. Images are subsequently processed for contrast and brightness and analyzed with imaging software. Higher magnification images (not shown here) can be accomplished by using a fluorescence stereomicroscope equipped with a 50 W mercury lamp. In this scenario, selective excitation of GFP is produced through a D425/60 band-pass filter and 470 DCXR dichroic mirror. Emitted fluorescence are captured and processed as described above. Images A and B demonstrate GFP transgene expression following adenoviral delivery to the brain. Image C demonstrates Ad-CMV-GFP delivery to the liver via portal vein cannulation. See color insert for color version of this figure. (Images courtesy of Anticancer, Inc., Ref. 18.)

nously delivered genes and to be used to monitor gene therapy (19). This new breed of fluorescent probe is dependent upon the close proximity of multiple near-infrared fluorochromes (NIRF), Cy5.5, when bound to a synthetic graft copolymer consisting of a cleavable backbone [partially methoxy poly(-ethylene glycol)-modified poly-L-lysine] (43). When placed in close proximity, a pair of these fluorochromes will ''quench'' each other and, therefore, not be detectable. Upon enzymatic cleavage of the backbone with a protease that has lysine-lysine specificity, the fluorochromes are spatially dissociated and will begin to fluoresce.

Because tumor progression and angiogenesis necessarily elaborate certain proteases, these clever or ''smart'' biocompatible autoquenched near-infrared fluorescent probes can be used to detect tumors that are known to upregulate certain proteases. Cathepsins B and H are tumor proteases that have lysine-lysine specificity and have been shown to activate this fluorescent probe in tumor xenografts. Other known tumor-enhanced proteases, Cathepsin D and matrix metalloprotei-nase-2 (MMP-2), which are dependent on other specific pep-tide sequences for their action, can also activate this probe if the NIR fluorochromes are attached to the backbone via Cathepsin D-sensitive or MMP-2-sensitive sequences; these enzyme-specific probes have been demonstrated in living subjects (19,44).

Bioluminescent optical systems are increasingly being used in the study of living subjects because of their inherently low background (no excitation irradiation needed that would otherwise cause background autofluorescence). The most commonly used bioluminescence reporter gene is the Firefly luciferase gene, Fluc, which encodes a 550 amino acid, 61-kDa monomeric protein (FL) derived from Photinus pyralis, the North American firefly (36). Photon emission is achieved through oxidation of its native substrate, D-Luciferin [D-( - )-2-(6'-hydroxy-2'-benzothiazolyl) thiazone-4-carboxylic acid] into oxyluciferin in a reaction that requires ATP, Mg2+, and O2. The reaction produces a broad spectral emission that peaks at 560 nm. A number of modifications to the gene since its discovery has facilitated its use in mammalian tissues, which include amino acid substitutions that red-shift the emissions peak above 600 nm, optimized mammalian codon language, removal of a peroxisome targeting site for increased expression levels, and cytoplasmic localization (21,45). Once produced, luciferase does not require post translational processing for enzymatic activity, and it can immediately function as a genetic reporter. Examples of the use adenoviral-mediated firefly luciferase gene delivery are given in Figs. 6 and 7. Figure 6 is an example of cardiac gene therapy and Fig. 7 is an example of how tissue-specific transgene expression can be achieved using tissue-specific promoters in living subjects.

A second bioluminescence system utilizing the Renilla luciferase gene (Rluc) that encodes a 36-kDa monomeric protein (RL), has recently been tested in small rodents (15). Its peak emission displays a blue-green bioluminescence at 480 nm when Renilla luciferase interacts with its substrate, coelen-terazine. In comparison to FL, RL does not need cofactors or ATP to oxidize its substrate, and therefore it will be less taxing to the cell in which it is expressed. It also has much more rapid kinetics in terms of light production so that it can potentially be used simultaneously with FL through the injection of both substrates and multiple time-point imaging (15).

Comparing the technical and practical aspects of fluorescent and bioluminescence systems reveals substantial differences, which are briefly mentioned here. Fluorescence imaging benefits greatly from relatively high photon yield from its proteins and dyes so that, in some cases, living subjects can be imaged with conventional photographic equipment rather than the more expensive, cooled CCD cameras. In comparison, the photon yield of bioluminescence systems is significantly lower, and imaging of living subjects always requires a cooled CCD camera. Fluorescence imaging can potentially be used to image simultaneous signals of different colors, whereas multiplexing different and simultaneous signals in bioluminescence imaging is difficult, owing to the differential enzyme kinetics and time-to-peak photon flux among varying luciferases.

On the other hand, bioluminescence imaging has certain advantages over the fluorescence systems. For example, autofluorescence, a source of noise in the image, is significantly less in bioluminescence for reasons discussed earlier. Also, bioluminescence reporters can be utilized as soon as they are synthesized. In contrast, the fluorescent proteins usually have a requisite period, some substantially longer than others, of posttranslational processing and maturation prior to their function as a fluorescent reporter (37). In some cases, where temporal resolution is an important issue in the monitoring of gene therapy, the use of fluorescence techniques may be limiting in this regard. Bioluminescence proteins also take some finite time to mature, but anecdotal evidence suggests that it is not as long as the fluorescent proteins; future studies are certain to address this issue.

Imaging with bioluminescence techniques permits rapid, repetitive, or prolonged imaging periods so long as the required substrates and cofactors are available. The physical phenomena of photobleaching, a property seen in many fluorescent proteins where the fluorochrome is permanently extinguished after light excitation or ultraviolet light exposure, makes imaging difficult if prolonged excitation periods or rapid repetitive imaging is needed; repeat imaging can only take place once fresh fluorescent protein has been generated. Photobleaching is not an issue in bioluminescence imaging. Having stated the above, no formal comparison has yet been made between fluorescence and bioluminescence using the same animal model, fusion reporter/individual reporters, and the same CCD camera. These studies should help to better define which is the more ideal reporter.

B. Radionuclide-Imaging Reporter Systems

Currently, PET or Gamma/SPECT reporter genes encode either a receptor, enzyme, or ion pump, which bind a radiolabeled ligand, interact with a radiolabeled substrate, or facilitate intracellular translocation of ionic radioisotopes, respectively. The reporter gene product is designed to sequester the

CONTROL (DAY 2) RLU/min

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