WO2004038039A2 - Optical imagiong of renilla luciferase reporter gene expression in living organisms - Google Patents

Abstract

Methods for detecting and localizing Renilla luciferase (RL) bioluminescence originating from an animal are disclosed. Also disclosed are methods for targeting RL light emission to selected regions, as well as for tracking entities within the animal.

Description

OPTICAL IMAGING OF RENILLA LUCIFERASE REPORTER GENE EXPRESSION IN

LINING ORGANISMS

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application claims the benefit of U.S. Provisional Application, Ser. No. 60/420,419, filed October 21, 2002, the content of which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Repetitive monitoring of reporter gene expression in intact living animals is crucial for many applications, including cell trafficking, gene therapy studies, and transgenic models (Ray, P., et al. (2001) Semin. Nucl. Med. 31, 321 330). Noninvasive, real time analysis of molecular events in intact living mammals is an active area of current research (See, e.g., Bremer, C. & Weissleder, R. (2001) Acad. Radiol. 8, 15 23). Several imaging technologies and new reporter genes are being studied for noninvasive imaging and quantitation of gene expression in living subjects. Some of the imaging modalities and established reporter genes include single photon emission computed tomography (SPECT) using Herpes Simplex Virus Type I thymidine kinase HSNl-tk, Somatostatin Type 2 receptor, and Sodium/Iodide Symporter as reporter genes. Positron emission tomography (PET) using HSNl-tk and Dopamine Type 2 Receptor as reporter genes, MRI with various reporter genes, and optical imaging approaches with fluorescence and bioluminescent reporter genes have also been studied. A detailed review of reporter gene approaches for use in living subjects can be found in Ray et al., supra).

For many applications, it would be very useful to have multiple reporter genes. Separate reporter genes can be used with different modalities (e.g., PET, MRI), but would lack convenience and high throughput, and images would be more difficult to co-register and quantitate.

Accordingly, there is a need for an improved method of optical imaging of a reporter gene that can be used in intact organisms.

SUMMARY OF THE INVENTION

In one embodiment, the invention includes a noninvasive method for detecting the localization of a cell within an animal, comprising administering to the animal a transformed cell expressing Renilla luciferase ("RL"), after a period of time in which the transformed cell can achieve localization in the animal, administering coelenterazine to the animal, immobilizing the animal within the detection field of a photodetection device, and detecting light emission from RL localized in the animal. In another embodiment, the invention includes a noninvasive method for detecting the localization of an entity within an animal, comprising administering to the animal an entity conjugated to RL protein, after a period of time in which the conjugated entity can achieve localization in the animal, administering coelenterazine to the animal, immobilizing the animal within the detection field of a photodetection device, and detecting light emission from RL localized in the animal.

In another embodiment, the invention includes a noninvasive method for detecting the level of expression of a gene of interest in an animal over time, comprising operably fusing a nucleic acid encoding RL to the gene of interest, administering the fused gene to the animal, administering coelenterazine to the animal, immobilizing the animal within the detection field of a photodetection device, and measuring the level of light emission from RL in the animal with the photodetection device at selected time intervals sufficient to detect changes in the level of light emission in the animal over time.

In another embodiment, the invention includes a method for detecting a promoter- induction event in an animal, comprising administering coelenterazine to a transgenic animal having a promoter responsive to the event and a heterologous gene encoding RL under the control thereof, triggering the event in the transgenic animal, and measuring with a photodetector device light emission through opaque tissue from the RL in the transgenic animal.

In another embodiment, the invention includes a noninvasive method for detecting the localization of a gene therapy vector within an animal, comprising operably fusing a nucleic acid encoding RL into the gene therapy vector, administering the fused gene therapy vector to the animal, after a period of time in which gene therapy vector can achieve localization in the animal, administering coelenterazine to the animal, immobilizing the animal within the detection field of a photodetection device, and detecting light emission from RL localized in the animal.

In another embodiment, the invention includes a method for detecting two independent promoter-induction events in an animal, comprising providing a transgenic animal having a first promoter responsive to the first event and a heterologous gene encoding RL under the control thereof and a second promoter responsive to the second event and a heterologous gene encoding FL under the control thereof, administering coelenterazine to the animal, administering D-luciferin to the animal, triggering the first event in the transgenic animal, triggering the second event in the transgenic animal, and measuring with a photodetector device light emission through opaque tissue in the transgenic animal.

These and other objects and features of the invention will be more fully appreciated when the following detailed description of the invention is read in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the kinetics of light production with C6-Rluc and C6 cell extracts exposed to coelenterazine. The graph shows a peak signal within the first 10 seconds that goes down steadily for the next 10 minutes. The values plotted were integrated every 10 seconds. Control C6 cells do not show any significant signal. The values are normalized to mg of total protein. The error bars represents standard error of mean (SEM) of triplicates.

FIG. 2 is a graph showing the effects of coelenterazine dose on measured light from C6-Rluc and control C6 lysates. A dose range of 0.1 to 50 μg/ml coelenterazine produces a maximum of 9.6 x 10⁵ + 1.1 x 10⁴ RLU/mg, as measured in the luminometer. A near linear increase with dose is observed between 0.1 to 10 μg/ml, with some plateauing between 10 to 50 μg/ml. Control C6 cell lysates do not show any significant signal. The signal from C6-Rluc cell lysates is significantly different (P < 0.05) from control with a dose as low as 0.1 μg/ml. The values are from triplicate wells normalized to mg protein. The error bar represents SEM.

FIG. 3 is a bar graph showing the crossreactivity of D-luciferin and coelenterazine with FL in cell culture. (A) The cells with C6-Rluc show bioluminescence with coelenterazine (CL) and not with D-luciferin (D-L). (B) The C6-Fluc cells showed bioluminescence with D-luciferin, whereas coelenterazine produces no significant signal from these cells. Control cells, untreated with substrate, show negligible signal in both. All RLU values are normalized to micrograms of total protein. The error bar represents the SEM for triplicate wells.

FIG. 4 shows that RL bioluminescence from C6-Rluc cells is present in various tissues in living mice. (A) The C6-Rluc cells (1 x 10⁶) were injected via tail-vein and coelenterazine was tail-vein injected 90 minutes later. The bioluminescence seen represents the thorax region of the mouse, where C6-Rluc cells are trapped in the lungs. (B) C6-Rluc cells (1 x 10⁶) were implanted in the peritoneum of a different mouse and coelenterazine was tail-vein injected immediately. Bioluminescence is seen only from the intraperitoneal region. "R" and "L" represent the right and left side of the mouse resting in a supine position.

FIG. 5 is a bar graph showing that RL bioluminescence from C6 Rluc cells in living mice is dependent on the dose of coelenterazine injected. A dose range of coelenterazine from 0.07 to 3.5 mg/kg body weight was injected via tail vein in two mice subcutaneously implanted with C6 Rluc cells. The region-of-interest signal increases as a function of higher coelenterazine dose. The error bar represents mean SEM.

FIG. 6 shows crossreactivity of RL for D-luciferin and FL for coelenterazine in living mice. Both C6-Fluc (A) and C6-Rluc (B) cells were implanted s.c. at right forearm and left forearm sites, respectively, in the same mouse with control C6 cells (C) implanted in the right thigh region. Injection of D-luciferin via tail-vein in the mouse I shows bioluminescence from site A and minimal signal from the B and C sites. Injection of coelenterazine via tail-vein in mouse II produces bioluminescence from site B but minimal signal from the A or C sites. R and L represent the right and left side of the mouse resting in supine position.

FIG. 7 shows the kinetics of light production from mice carrying s.c. C6-Fluc and C6-Rluc cells after simultaneous tail-vein injection of both D-luciferin and coelenterazine. A mouse was injected s.c. with C6-Fluc (A), C6-Rluc (B), and C6 control cells (C) on right forearm, left forearm, and right thigh regions, respectively. Simultaneous injection of both D-luciferin and coelenterazine mixture via tail- vein shows bioluminescence from both the sites simultaneously but with distinct kinetics. A series of images at 2-min intervals is shown from the same mouse. Each image represents a scan time of one minute. The signal from C6-Rluc cells (B) peaks early and is near extinguished within 10 minutes. Bioluminescence from C6-Fluc cells (A) shows a relatively strong signal beyond 10 minutes. The region of control cells (C) does not show any significant bioluminescence. R and L represent the right and left side of the mouse resting in supine position.

FIG. 8. Comparison of signals in different cell lines using two different Renϊlla reporter genes. The graph shows four different cell lines used for comparing signal produced from hRL: coelenterazine and RL:coelenterazine as measured in a luminometer. The cells were transiently transfected with pCMV -Rluc/pCMV-hRluc and the cell lysates assayed 24 hr post transfection with the substrate coelenterazine, to compare the luciferase activity of two groups of transfected cells. All the cell lines show significant (P<0.05) enhancement in signal from cells transfected with pCMV-hRluc plasmid as compared to the pCMV-RZwc plasmid. Signals from the control cell lysates are negligible. Note the y-axis is in log scale. The values are normalized to μg of total protein/second and for transfection efficiency (β-gal activity). The error bars (not seen) represents standard error of mean (SEM) of triplicates.

FIG. 9. Effects of coelenterazine dose on measured light from Cβ-hRluc, C6-Rluc and control C6 cell lysates. C6 cells were transiently transfected with pCMV-tzRZwc, pCMV-RZwc, or mock transfected. Cell lysates were prepared and equal volume of lysates (5μl) were mixed with increasing concentration of coelenterazine. The RLU was measured in the luminometer. The signals from C6-hRluc cell lysates are -250 fold higher than C6-Rluc lysates at any given dose. A near linear increase with dose is observed between 0.01-1 μg/ml of coelenterazine with C6-hRluc lysates. Control C6 cell lysates do not show any significant signal. The y-axis is in log scale. The values are from triplicate wells normalized to μg protein/second/β-gal activity. The error bars (not seen) represent SEM of triplicates.

FIG. 10. RT-PCR and Western blotting of C6/C6-Rluc/C6-hRluc cell extracts. Total RNA from C6-hRluc cells and C6-Rluc cells was isolated and equal amounts (200ng) were analyzed by reverse transcription PCR as described below. The 203bp RT-PCR products were separated in 2% agarose gel stained with ethidium bromide, (a) An inverted image of the gel shows a higher amount of RT-PCR product from C6-hRluc mRNA as compared to equal volume (lOμl) C6-Rluc cell mRNA [Lane 1, molecular weight markers (lOObp); lane 2, C6-hRluc; lane 3, C6-Rluc; lane 4, C6 control], (b) The total protein were extracted from C6, C6-Rluc and C6-hRluc cells using Laemmli's sample buffer. Equal amount (10 μg) of cellular protein were loaded in each lane. A higher expression of hRL is observed as compared to RL as seen by the 35 kDa band in the western blot treated with anti- Renilla antibody [lane 1, C6 control cells; lane 2, C6-hRluc; lane 3, C6-RZwc].

FIG. 11. RL bioluminescence in living mice is dependent on the dose of coelenterazine injected. Coelenterazine doses ranging from 0.07-2.1 mg/kg body weight were injected via tail vein in duplicate sets of CDl mice subcutaneously implanted with C6- hRluc and C6 control cells. The ROI signal increases as a function of higher coelenterazine dose from the C6-hRluc site. The error bar represents mean ± SEM.

FIG. 12. Optical imaging of mice carrying both C6-hRluc and C6-Rluc cells at two different s.c. sites. A mouse was injected subcutaneously with C6-Rluc, C6-hRluc and C6 control cells on left forearm, right forearm and right thigh regions respectively. Injection of coelenterazine (0.7 mg/kg body weight) via tail-vein shows bioluminescence from both the C6-Rluc and C6-hRluc sites simultaneously with distinct kinetics, (a) An image recorded from a mouse using a one minute scan with the cooled CCD camera demonstrating the higher signal from C6-hRluc as compared to C6-Rluc cells and background signal from the site of C6 cells. R and L represent the right and left side of the mouse resting in supine position, (b) Data demonstrating significantly higher and prolonged signal from the C6-hRluc site as compared to the C6-Rluc site. Note the y-axis is in log scale.

FIG. 13. Cell Culture and RT-PCR of C6/C6-Fluc/C6-hRluc cell extracts. Total RNA from C6-hRluc cells and C6-Fluc cells were isolated and equal amounts analyzed by reverse transcription PCR as described below. The ~203bp RT-PCR products were separated in 2% agarose gel stained with ethidium bromide, (a) An inverted image of the gel shows a higher amount of RT-PCR product from C6-hRluc mRNA as compared to C6-Fluc cell mRNA [Lane 1, C6 control; lane 2, C6-Fluc; lane 3, molecular weight markers (lOObp); lane 4, C6-hRluc]. (b) The cell culture data shows -17 fold difference in the signal from C6- hRluc as compared to C6-Fluc cell lysates when checked in the luminometer. Data represented as RLU/μg protein/sec/β-gal activity and the error bar represents mean ± SEM. Note the y-axis is in log scale.

FIG. 14. Optical imaging of mice carrying subcutaneous C6-hRluc, C6-Fluc cells, and C6 cells after tail-vein injection of a mixture of coelenterazine and D- Luciferin. A mouse was injected subcutaneously with C6-Fluc, C6-hRluc and C6 control cells on right forearm, left forearm and right thigh regions respectively. Injection of a coelenterazine and D-luciferin mixture via tail-vein shows bioluminescence from both the sites simultaneously with distinct kinetics. R and L represent the right and left side of the mouse in the supine position.

FIG. 15. Optical imaging of mice with C6-hRluc and C6-Fluc cells trafficked to lungs. A mouse was injected with a mixture of one million C6-Fluc and one million C6- hRluc cells via tail-vein. The cells were allowed to traffic into the lungs by waiting for 2 hours, (a) Separate injection via tail-vein of coelenterazine (5.7 mg/kg) and two hours later with i.p. injection of D-luciferin (150 mg/kg) shows different levels of signal from the two cell populations in the same mouse. The animal injected with coelenterazine (1; left panel) shows a significantly (P<0.05) higher signal from the C6-hRluc population located in the lung region as compared to the same animal injected with D-Luciferin after 2 hours (2; right panel), (b) Mean data from three mice demonstrating significantly higher (P<0.05) signal from C6-hRluc as compared to the C6-Fluc cells in the lung region. Note the y-axis is in log scale. Error bars represent s.e.m.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices and materials are now described.

All patents, patent applications, journal articles and other publications mentioned in this specification are incorporated herein in their entireties by reference for the purpose of describing and disclosing, for example, compositions and methodologies that are described in the publications which might be used in connection with the presently described invention. The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.

One aspect of the present invention is a method for detecting expression of a reporter gene in an intact organism comprising the steps of:

(1) introducing a Renilla luciferase reporter gene to the organism under control of a promoter that is operatively linked to the reporter gene;

(2) inducing expression of the reporter gene; and

(3) detecting the expression of the reporter gene by detecting the light produced by the enzymatic activity of the protein encoded by the reporter gene with a cooled charge coupled device camera.

The enzyme Renilla luciferase (RL), purified from sea pansy (Renilla reniformis), is a bioluminescent compound that displays blue green bioluminescence upon mechanical stimulation. It is widely distributed among coelenterates, fishes, squids, and shrimps (J.W. Hastings, (1996) Gene 173, 5-11). It has been cloned and sequenced by Lorenz et al. (1991) Proc. Natl. Acad. Sci. USA 88, 4438-4442, and used as marker of gene expression in bacteria, yeast, plant, and mammalian cells (W.W. Lorenz et al., (1996) J. Biolumin. Chemilumin. 11, 31-37). The enzyme RL catalyzes coelenterazine oxidation leading to bioluminescence.

Coelenterazine consists of an imidazolopyrazine structure, {2-(p-hydroxybenzyl)-6- (p-hydroxyphenyl)-8-benzylimidazo-[l,2-a]pyrazin-3-(7H)-one} that releases blue light across a broad range, peaking at 480 nm upon oxidation by RL in vitro (J.C. Matthews et al., (1977) Biochemistry 16, 5217-5220).

Other bioluminescence systems exist. One well-characterized bioluminescent enzyme is known as firefly luciferase ("FL"), because it was isolated from the lightning bug or the firefly. Like RL, FL will also produce light in the presence of its substrate, which in this case is D-luciferin. FL has been used in live animal systems, and the resulting light production imaged. See, for example, U.S. Pats. Nos. 5,650,135 and 6,217,847, the entire contents of which are incorporated by reference herein. Accordingly, another aspect of the present invention is the use of Renilla luciferase reporter gene together with firefly luciferase reporter gene in the same organism simultaneously. This is because the light emitted by the two bioluminescence systems is at different wavelengths, with the Renilla luciferase generating a flash of blue luminescence with a wavelength centering at 482 nm, and the firefly luciferase giving a flash of green light at 562 nm.

Firefly luciferase is a 61 kDa single subunit protein that catalyzes D-luciferin to produce oxyluciferin in the presence of oxygen, cofactors, Mg²⁺, and ATP to yield green light. It should be noted that the two luciferases and the two substrates coelenterazine and D- luciferin are structurally unrelated.

Accordingly, another aspect of the present invention is a method for imaging two reporter genes comprising the steps of:

(1) providing Renilla luciferase reporter gene to cells of an organism under control of a first promoter that is operatively linked to the Renilla luciferase reporter gene;

(2) providing a firefly luciferase reporter gene to the organism under the control of a second promoter that is operatively linked to the firefly luciferase reporter gene;

(3) inducing expression of the Renilla luciferase reporter gene and of the firefly luciferase reporter gene;

(4) detecting the expression of the Renilla luciferase reporter gene by detecting the light produced by the enzymatic activity of the Renilla luciferase enzyme and emitted at about 480 nm with a cooled charge coupled device camera; and

(5) detecting the expression of the firefly luciferase reporter gene by detecting the light produced by the enzymatic activity of the firefly luciferase enzyme and emitted at about 562 nm with a cooled charge coupled device camera to differentially detect the expression of the Renilla luciferase reporter gene and the firefly luciferase reporter gene.

The use of Renilla luciferase and firefly luciferase provides a mechanism in which two independent optical signals can be used for measurement of the location, magnitude, and persistence of expression of two different genes.

The method can be used for detecting expression of reporter genes in intact organisms, as well as in tissues and organs being maintained in culture or in tissue slices or other ex vivo or in vitro situations.

The invention includes entities which have been modified or conjugated to include RL and which may take the form of, for example, molecules, macromolecules, particles, microorganisms, or cells. The methods used to conjugate RL to an entity depend on the nature of the entity. Exemplary conjugation methods are discussed in the context of the entities described below.

Small molecules. Small molecule entities which may be useful in the practice of the present invention include compounds which specifically interact with a pathogen or an endogenous ligand or receptor. Examples of such molecules include, but are not limited to, drugs or therapeutic compounds, toxins, growth factors, cytokines, and bioactive peptides.

Conjugations are typically chemical in nature, and can be performed by any of a variety of methods known to those skilled in the art. The small molecule entity may be synthesized to contain RL, so that no formal conjugation procedure is necessary. Alternatively, the small molecule entity may be synthesized with a reactive group that can react with the light generating moiety, or vice versa.

Small molecules conjugated to RL may be used either in animal models of human conditions or diseases, or directly in human subjects to be treated. For example, a small molecule which binds with high affinity to receptor expressed on tumor cells may be used in an animal model to localize and obtain size estimates of tumors, and to monitor changes in tumor growth or metastasis following treatment with a putative therapeutic agent. Such molecules may also be used to monitor tumor characteristics, as described above, in cancer patients.

Macromolecules. Macromolecules, such as polymers and biopolymers, constitute another example of entities useful in practicing the present invention. Exemplary macromolecules include antibodies, antibody fragments, fusion proteins and certain vector constructs.

Antibodies or antibody fragments, purchased from commercial sources or made by methods known in the art (Harlow, et al., 1988, Antibodies: A Laboratory Manual, Chapter 10, pg. 402, Cold Spring Harbor Press), can be used to localize their antigen in a mammalian subject by conjugating the antibodies to RL, administering the conjugate to a subject by, for example, injection, allowing the conjugate to localize to the site of the antigen, and imaging the conjugate.

Antibodies and antibody fragments have several advantages for use as entities in the present invention. By their nature, they constitute their own targeting moieties. Further, their size makes them amenable to conjugation with RL, yet allows them to diffuse rapidly relative to, for example, cells or liposomes.

RL can be conjugated directly to the antibodies or fragments, or indirectly by using, for example, a secondary antibody. Direct conjugation can be accomplished by standard chemical coupling of RL to the antibody or antibody fragment, or through genetic engineering. Chimeras, or fusion proteins can be constructed which contain an antibody or antibody fragment coupled to a RL protein. (See, e.g., Casadei, et al., 1990, PNAS 87:2047- 2051).

Vector constructs by themselves can also constitute macromolecular entities applicable to the present invention. For example, a eukaryotic expression vector can be constructed which contains a therapeutic gene and a gene encoding RL under the control of a selected promoter (i.e., a promoter which is expressed in the cells targeted by the therapeutic gene). Expression of RL, assayed using methods of the present invention, can be used to determine the location and level of expression of the therapeutic gene. This approach may be particularly useful in cases where the expression of the therapeutic gene has no immediate phenotype in the treated individual or animal model.

Viruses. Another entity useful for certain aspects of the invention are viruses. As many viruses are pathogens which infect mammalian hosts, the viruses may be conjugated to RL and used to study the initial site and spread of infection. In addition, viruses labeled with RL may be used to screen for drugs which inhibit the infection or the spread of infection.

A virus may be labeled indirectly, either with an antibody conjugated to RL, or by, for example, biotinylating virions (e.g., by the method of Dhawan, et al., 1991, J. Immunol. 147(1): 102) and then exposing them to streptavidin linked to RL.

Alternatively, virions may be labeled directly with RL, using, for example, the methods of Fan, et al., 1992, J. Clin. Micro. 30(4):905. The virus can also be genetically engineered to express RL. The genomes of certain viruses, such as herpes and vaccinia, are large enough to accommodate genes as large as the Rluc or hRluc genes used in experiments performed in support of the present invention.

Labeled virus can be used in animal models to localize and monitor the progression of infection, as well as to screen for drugs effective to inhibit the spread of infection.

Particles. Particles, including beads, liposomes and the like, constitute another entity useful in the practice of the present invention. Due to their larger size, particles may be conjugated with a larger number of RL molecules than, for example, can small molecules. This results in a higher concentration of light emission, which can be detected using shorter exposures or through thicker layers of tissue. In addition, liposomes can be constructed to contain an essentially pure targeting moiety, or ligand, such as an antigen or an antibody, on their surface.

Cells. Cells, both prokaryotic and eukaryotic, constitute another entity useful in the practice of the present invention. Like particles, cells can be loaded with relatively high concentrations of RL, but have the advantage that the RL can be provided by, for example, a heterologous genetic construct used to transfect the cells. In addition, cells can be selected that express "targeting moieties", or molecules effective to target them to desired locations within the subject. Alternatively, the cells can be transfected with a vector construct expressing an appropriate targeting moiety.

Bacterial cells constitute effective entities. For example, they can be easily transfected to express a high levels of RL, as well as high levels of a targeting protein. In addition, it is possible to obtain E. coli libraries containing bacteria expressing surface-bound antibodies which can be screened to identify a colony expressing an antibody against a selected antigen (Stratagene, La Jolla, Calif.). Bacteria from this colony can then be transformed with a second plasmid containing an Rluc or hRluc gene, and transformants can be utilized in the methods of the present invention, as described above, to localize the antigen in a mammalian host.

Pathogenic bacteria can be conjugated to RL and used in an animal model to follow the infection process in vivo and to evaluate potential anti-infective drugs, such as new antibiotics, for their efficacy in inhibiting the infection.

Eukaryotic cells are also useful as entities in aspects of the present invention. Appropriate expression vectors, containing desired regulatory elements, are commercially available. The vectors can be used to generate constructs capable of expressing RL in a variety of eukaryotic cells, including primary culture cells, somatic cells, lymphatic cells, etc. The cells can be used in transient expression studies, or, in the case of cell lines, can be selected for stable transformants.

Expression of RL in transformed cells can be regulated using any of a variety of selected promoters. For example, if the transformed cells are to be targeted to a site in the subject by an expressed ligand or receptor, a constitutively-active promoter, such as the CMV or SV40 promoter may be used. Cells transformed with such a construct can also be used to assay for compounds that inhibit light generation, for example, by killing the cells.

Alternatively, the transformed cells may be administered such they become uniformly distributed in the subject, and express RL only under certain conditions, such as upon infection by a virus or stimulation by a cytokine. Promoters that respond to factors associated with these and other stimuli are known in the art.

Tumor cell lines transformed as above, for example, with a constitutively-active promoter, may be used to monitor the growth and metastasis of tumors. Transformed tumor cells may be injected into an animal model, allowed to form a tumor mass, and the size and metastasis of the tumor mass monitored during treatment with putative growth or metastasis inhibitors.

Tumor cells may also be generated from cells transformed with constructs containing regulatable promoters, whose activity is sensitive to various infective agents, or to therapeutic compounds.

Cell Transformation. Transformation methods for both prokaryotic cells and eukaryotic cells are well known in the art (Sambrook, et al., 1989, In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Vol. 2). Vectors containing the appropriate regulatory elements and multiple cloning sites are widely commercially available (e.g., Stratagene, La Jolla, Calif., Clontech, Palo Alto, Calif.).

In the case of "targeted" entities, that is, entities which contain a targeting moiety— a molecule or feature designed to localize the entity within a subject or animal at a particular site or sites, localization refers to a state when an equilibrium between bound, "localized", and unbound, "free" entities within a subject has been essentially achieved. The rate at which such an equilibrium is achieved depends upon the route of administration. For example, a conjugate administered by intravenous injection to localize thrombi may achieve localization, or accumulation at the thrombi, within minutes of injection. On the other hand, a conjugate administered orally to localize an infection in the intestine may take hours to achieve localization.

Alternatively, localization may simply refer to the location of the entity within the subject or animal at selected time periods after the entity is administered. For example, entities conjugated to RL may be administered orally and their spread followed as a function of time. In this case, the entity can be "localized" immediately following the oral introduction, inasmuch as it marks the initial location of the administered bacteria, and its subsequent spread or recession (also "localization") may be followed by imaging.

In a related aspect, localization of, for example, injected tumors cells expressing RL, may consist of the cells colonizing a site within the animal and forming a tumor mass.

In all of the above cases, a reasonable estimate of the time to achieve localization may be made by one skilled in the art. Furthermore, the state of localization as a function of time may be followed by imaging the light-emitting conjugate according to the methods of the invention.

An important aspect of the present invention is the selection of a photodetector device with a high enough sensitivity to enable the imaging of faint light from within a mammal in a reasonable amount of time, preferably less than about 30 minutes, and to use the signal from such a device to construct an image.

One type of photodetector device suitable for practice of the present invention are devices which achieve sensitivity by reducing the background noise in the photon detector, as opposed to amplifying the photon signal. Noise is reduced primarily by cooling the detector array. The devices include charge coupled device (CCD) cameras referred to as cooled CCD cameras. In the more sensitive instruments, the cooling is achieved using, for example, liquid nitrogen, which brings the temperature of the CCD array to approximately -120. degree. C. A particularly sensitive cryogenic CCD camera is the "TECH 512", a series 200 camera available from Photometries, Ltd. (Tucson, Ariz.). CCD cameras with intensifiers, such as microchannel intensifiers, may also be used. Other suitable photodetection devices are known in the art and are described in, for example, U.S. Pat. No. 5,650,135.

Signals generated by photodetector devices which count photons need to be processed by an image processor in order to construct an image which can be, for example, displayed on a monitor or printed on a video printer. Such image processors are typically sold as part of systems which include the sensitive photon-counting cameras described above, and accordingly, are available from the same sources (e.g., Photometries, Ltd.).

The image processors are usually connected to a personal computer, such as an IBM- compatible PC or an Apple Macintosh (Apple Computer, Cupertino, Calif.), which may or may not be included as part of a purchased imaging system. Once the images are in the form of digital files, they can be manipulated by a variety of image processing programs well known in the art.

Imaging hardware and software suitable for practice of the present invention are available from Xenogen Corp. (Alameda, California).

The invention is illustrated by the following Examples. These examples are for illustrative purposes only and are not intended to limit the invention.

Example 1 Optical imaging of Renilla luciferase reporter gene expression in living mice

Abbreviations

CCD, charged coupled device; FL, firefly luciferase enzyme/protein; RL, Renilla luciferase enzyme/protein; hRL, synthetic Renilla luciferase enzyme/protein; Rluc, Renilla luciferase reporter gene; hRluc, synthetic Renilla luciferase reporter gene; SPB, sodium phosphate buffer; RLU, relative light units; ROI, region of interest. Materials and Methods

Cell Lines, Culture Conditions, and Transfection Procedures. C6 rat glioma cells were maintained in glucose deficient Minimum Eagle's Medium (MEM) supplemented with 1% penicillin-streptomycin, 1% L-glutamine, and 5% FCS. HeLa (human cervical carcinoma) cells, N2a (mouse neuroblastoma) cells, and 293 (human kidney) cells were maintained in DMEM supplemented with 1% penicillin-streptomycin and 10% FCS, whereas human prostrate adenocarcinoma, PC-3, cells were maintained in RPMI medium 1640 supplemented with 1% antibiotics and 5% FCS.

For assessment of Rluc expression in various cell types, each cell type described above was plated in 12-well plates (Costar) and transfected with pCMV-RZwc plasmid (Promega), using SuperFect Transfection Reagent (Qiagen). Mock transfected cells were used as control. Cells were lysed in lysis buffer 48 h post transfection, and biochemical studies were carried out using a luminometer as described below.

For additional cell culture studies, C6 cells were plated in 12 well plates and transiently transfected with either pCMN-FZwc (provided by C. H. Contag, Stanford University, Stanford, CA) or pCMV-RZwc plasmid, or mock transfected using SuperFect Transfection Reagent. These C6 cells transiently expressing Flue and Rluc are referred to as C6-Fluc and C6-Rluc, respectively. Bioluminescent signals from intact cells were detected directly by a cooled CCD camera. C6 cells were also grown in 100-mm plates (Costar), and transfected with pCMV-RZwc under similar conditions. They were collected by trypsinization 48 h post transfection, washed with PBS, counted, and 1 x 10⁶ cells (in 100 μl PBS) were used for in vivo studies as described below.

Preparation of Coelenterazine and D-luciferin. Coelenterazine (also known as "native coelenterazine"), a substrate for Renilla luciferase enzyme/protein ("RL"), was purchased from Biotium (Hayward, CA). The compound (2 mg/ml) was dissolved in methanol. Further dilutions were made in 50 mM sodium phosphate buffer (SPB), pH 7. D- luciferin firefly potassium salt, the substrate for firefly luciferase enzyme/protein ("FL"), was purchased from Xenogen (Alameda, CA). A 30 mg/ml stock in PBS was filtered through 0.22-μm filters before use.

Luminometer Measurements. All bioluminescent assays were performed in a TD 20/20 luminometer (Turner Designs, Sunnyvale, CA). Twenty microliters of crude and clarified cell lysates obtained from C6-Rluc cells and mock transfected C6 control cells were mixed with 100 μl of coelenterazine solution (50 μg/ml) prepared in SPB, pH 7.0. The reaction was measured over 10 min, every 10 s in the luminometer. The protein content of the cell lysates were determined with Bio Rad protein assay system (Bio-Rad) in a Beckman DU-50 spectrophotometer (Beckman Instruments) and the luminescence results reported as relative light units ("RLU") per milligram of protein.

Coelenterazine Dose Studies in Vitro. Twenty microliters of C6-Rluc and mock transfected C6 control cell lysates were mixed with 100 μl of coelenterazine prepared at various concentrations (0.1, 1.0, 10, 50, 100, and 200 μg/ml in SPB) and the dose-dependent RLU were recorded using a luminometer for 10 s. The lysates were collected from three separate wells for each dose and the bioluminescence was normalized to protein content.

Crossreactivity Studies in Cell Culture. To check the crossreactivity of RL with D- luciferin and FL with coelenterazine, C6-Fluc, and C6-Rluc cells were treated with each substrate and analyzed directly by using the cooled CCD camera. Two 12- well plates, one plated with C6-Fluc and the other with C6-Rluc, were prepared. In each plate, to one row of three wells, 2 μg/ml of coelenterazine, and to the second row 150 μg/ml of D-luciferin was added. The last row consisted of mock transfected control C6 cells. The substrates were diluted in culture media. The plates were placed in the CCD imaging system (described below) and the images acquired for 1 min. The cells were lysed with lysis buffer and protein content from each well was determined. Results are reported as bioluminescence normalized to protein content.

Imaging and Quantification of Bioluminescence Data. The in vivo Imaging System (IVIS, Xenogen), consisting of a cooled CCD camera mounted on a light tight specimen chamber (dark box), a camera controller, a camera cooling system, and a Windows computer system, was used for data acquisition and analysis (Wu, J. C, et al. (2001) Mol. Ther. A, 297-306). Each 12 well plate sample or supine mouse was placed in the specimen chamber mounted with the CCD camera cooled to -120°C, with a field of view ("FOV") set at 25 cm above the sample shelf. The photon emission, transmitted from cell samples and mice was measured. The gray scale photographic images and bioluminescence color images were superimposed using the LIVINGJMAGE V. 2.11 software overlay (Xenogen) and IGOR image analysis software (V. 4.02 A, WaveMetrics, Lake Oswego, OR). A region of interest ("ROI") was manually selected over the signal intensity. The area of the ROI was ι 9 1 kept constant and the intensity was recorded as maximum [photons -s^" -cm^" -sr^" (steradian)] within a ROI.

Imaging RL Bioluminescence in Various Tissues. All animal handling was performed in accordance with University of California, Los Angeles, and Animal Research Committee guidelines. Three sets of CD-I mice, 4 weeks old (approximately 30 g; Charles River Breeding Laboratories) in duplicates, were anesthetized by i.p. injection of -40 μl of a ketamine and xylazine (4:1) solution. To check for background signal from animals not expressing Rluc, one set of mice was tail vein injected with 0.7 mg/kg body weight of coelenterazine. Of the two other sets, the first mouse set was injected with Q6-Rluc cells (1 x 10⁶ cells in 100 μl of PBS) directly into the peritoneal cavity and the second set had the same number of cells injected via tail vein. One hundred microliters of 0.36 mg/kg body weight of coelenterazine was injected immediately via tail vein to observe the bioluminescence from the peritoneum. A higher dose of 2.8 mg/kg body weight of coelenterazine was injected after 90 min of cell injection to the second set, to obtain, sufficient bioluminescence from deep tissues such as the liver and lung. A whole body image was acquired using the cooled CCD camera.

Effects of Coelenterazine Dose in Mouse Studies. A group of 4 week old CD-I mice were anesthetized followed by s.c. implantation of C6-Rluc cells (1 x 10⁶ cells in 100 μl of PBS) in the left forearm region and C6 (control cells) in the right thigh region. Different doses of coelenterazine (0.07, 0.36, 0.7, 1.4, 2.1, 2.8, and 3.5 mg/kg body weight) were injected via tail vein in duplicate mice. Bioluminescence was measured from both C6 control and C6-Rluc sites over a 10-min time period by using ten 1-min acquisition scans.

Substrate Crossreactivity Studies and Comparison of Flue and Rluc Expression in Living Mice. Four sets of anesthetized mice (three mice in each set) were injected at three sites: C6-Rluc into the left forearm, C6-Fluc into the right forearm, and C6 control cells into the right thigh region. To the first set, 100 μl of coelenterazine (0.7 mg/kg body weight) was injected via tail vein and the mice were scanned with fifteen 1-min scans using the cooled CCD camera. To the second set, 100 μl of D-luciferin (150 mg/kg body weight) was injected via tail vein and scanned with fifteen 1 min scans. To the third set, a mouse with implanted C6 cells was first injected with 100 μl of coelenterazine solution (0.7 mg/kg body weight) via tail vein and bioluminescence was recorded using the cooled CCD camera with a 1 min acquisition time. After 3 h, 100 μl of D-luciferin solution (150 mg/kg body weight) was injected again, via tail vein to the same mouse and an image obtained for 1 min. One mouse in each set was also imaged again by injecting each substrate i.p. instead of via tail vein.

To study the kinetics of light production from FL and RL in vivo, 200 μl of a mixture of D-luciferin (150 mg/kg body weight) and coelenterazine (0.7 mg/kg body weight; 1:1) was injected via tail vein to the fourth set of mice. Bioluminescence was measured using ten 1- min acquisition scans. Results:

Different Cell Lines Can Be Successfully Transfected with the pCMV-RZwc Plasmid. Cell lines from different tissue origins (C6, HeLa, N2a, 293, PC-3) were transiently transfected with the pCMV-RZwc plasmid to check the expression of Rluc. All cell lines shows significantly higher (P < 0.05) levels of gene expression compared with the mock transfected control cells as assessed by the luminometer by using triplicate samples (data not shown). Successful transfection in different cell lines indicates that Rluc can be expressed in different tissues.

Coelenterazine Induces Flash Kinetics Within the First 10 s in C6-Rluc Cell Lysates, Which Rapidly Decays with Time. We first determined the time kinetics of light production from RL with the substrate coelenterazine. Cell lysates from transiently transfected C6-Rluc cells were mixed with 50 μg/ml of coelenterazine and the bioluminescent emission was recorded in the luminometer. The light intensity is highest within the first 10 s of the reaction and drops significantly over the course of the next 10 min (FIG. 1). Mock transfected control C6 cells show a negligible bioluminescence (0.96 x 10³ + 0.14 x 10² RLU/mg).

Bioluminescence from C6-Rluc Cell Lysates Increases with Higher Doses of Coelenterazine. To determine the effects of coelenterazine dose on light yield, a study with coelenterazine and C6-Rluc cell lysates was performed using the luminometer. Coelenterazine as low as 0.1 μg/ml is able to produce detectable light with cell lysates (FIG. 2). An approximately linear relationship between the doses of coelenterazine (0.1 - 10 μg/ml) and signal intensity is observed. The peak signal is found with 50 μg/ml of coelenterazine within the first 10 s of reaction. With increasing doses (>50 μg/ml), there is a significant decrease in the signal that is probably due to absorption of light in the buffer that becomes yellow in color with higher concentration of coelenterazine (data not shown). The optimum dose of coelenterazine for further in vitro studies was chosen to be 50 μg/ml.

Coelenterazine and D-luciferin Do Not Exhibit Crossreactivity to FL or RL, Respectively, in Cell Culture. We next checked the crossreactivity of D-luciferin with C6- Rluc cells and coelenterazine with C6-Fluc cells directly in cell culture, using the cooled CCD camera. FIG. 3A shows data from the C6-Rluc cells with negligible bioluminescence when D-luciferin is added to the cell media or when mock transfected C6 cells without addition of substrate are imaged. C6-Rluc cells exposed to coelenterazine show a significantly higher signal (P < 0.01). Similarly, in the 0,6-Fluc cell plate (FIG. 3B), three wells treated with coelenterazine exhibited minimal signal with C6-Fluc cells as did control cells untreated with substrate, but cells exposed to D-luciferin show a significantly higher signal (P < 0.01).

C6-Rluc Cells Present in Various Tissues in Living Mice Can Be Imaged in the Cooled CCD Camera After Injection of Coelenterazine. To check the background signal from control mice, duplicate mice were tail vein injected with 0.7 mg/kg body weight of coelenterazine. They show a relatively low background bioluminescence of 2.78 x 10³ + 0.46 x 10³ maximum (photons-s^-cm^-sr^"1). To further check whether bioluminescence could be detected from various tissues, 1.0 x 10⁶ C6-Rluc cells suspended in 100 μl of PBS were injected via tail vein (for cell trafficking to liver and lungs) and into the peritoneal cavity in separate sets of animals. The bioluminescent signal is detected from the thorax region when coelenterazine is tail vein injected 90 min after cell injection (FIG. 4A). Earlier imaging showed cells initially trafficking to liver region (data not shown). The signal increases with time (peaking at -5 min) and subsides within 7 - 10 min after injection of coelenterazine. Sacrifice of mice and luminometer assessment of tissue homogenates revealed that more signal was present in the right vs. left lung (data not shown).

Bioluminescent signal is also detected from cells in the peritoneum after tail vein injection of coelenterazine (FIG. 4B). The peak signal was seen -3 min after injection of coelenterazine and retained for -10 - 12 min. C6-Rluc cells implanted s.c. show a peak signal at -1 min after tail vein injection of coelenterazine (described below). These results were consistent across two different mice.

RL Signal Enhances with Increasing Coelenterazine Dose in Living Mice. C6- Rluc cells (1 x 10⁶ cells in 100 μl PBS) were s.c. implanted into the left shoulder of a mouse while the mock transfected C6 cells were implanted in the right thigh. When the mouse is injected with coelenterazine (0.07 mg/kg body weight) via tail vein there is a detectable bioluminescence of -8.3 x 10³ + 0.15 x 10³ maximum (photons-s^"1 -cm^-sr^"1) from an ROI drawn over the site of implantation at the left shoulder area. This signal subsides within 5 min. The C6 control site at the right thigh region shows a signal of -3.1 x 10³ + 0.5 x 10³

1 9 1 maximum (photons-s^" -cm^" -sr^" ). There is a progressive increase in the bioluminescence from the implanted cells with increasing coelenterazine dose from 0.07 - 3.5 mg/kg body weight (FIG. 5). The duration of bioluminescence also increases with the dose of substrate (data not shown). We kept the dose in the range of 0.36 - 0.7 mg/kg body weight in further studies to minimize costs.

There Is Minimal Signal from C6-Rluc and C6-Fluc Cells Implanted Subcutaneously in Mice upon Tail Vein Injection of D-luciferin and Coelenterazine, Respectively. To check for any crossreactivity between the two proteins and substrates in vivo, C6 control, C6-Rluc, and C6-Fluc cells were implanted into the right thigh, left forearm, and right forearm, respectively, and tail vein injected coelenterazine was administered, followed later by D-luciferin. A significant level of bioluminescence was observed only from the C6-Fluc implanted site when D-luciferin was injected (FIG. 6, mouse I, site A), with background signal from C6 Rluc and control cell sites (FIG. 6, mouse I, sites B and C). Bioluminescence was seen only from the C6-Rluc site when coelenterazine was injected (FIG. 6, mouse II, site B). There was no sign of any crossreactivity with up to 15 minutes of repetitive scanning. Each substrate was also injected into the same mouse, 3 hours apart, giving sufficient time for the first signal from coelenterazine to dissipate completely, followed by D-luciferin injection, and also found a lack of crossreactivity (data not shown). These results are consistent across three different mice. Similar results are obtained when mice had substrates injected i.p., except that there is a delay in the time to peak bioluminescent signal (data not shown).

Bioluminescence in Living Mice Implanted with C6 Rluc and C6 Flue Shows Distinct Kinetics from Each of the Two Reporter Proteins. As shown above, RL and FL do not crossreact with each of their respective substrates in vitro or in vivo. Therefore, to determine the kinetics of light production when both substrates are injected simultaneously, a mixture of D-luciferin and coelenterazine was injected via tail vein into the same mouse implanted with both C6-Rluc and C6-Fluc at two s.c. forearm sites and control C6 cells at the thigh region (FIG. 7). It should be noted that the amount of coelenterazine injected was very low (0.7 mg/kg body weight) compared with D-luciferin (150 mg/kg body weight). The C6- Rluc implanted site (left forearm) showed a quick peak in the signal within 1 min, which consistently decreased over the 10 min period. On the other hand, the C6-Fluc site (right forearm) showed a progressive increase of the signal until approximately 3 to 4 min after tail vein injection, followed by a progressive decrease in the signal. There was strong bioluminescence from Q.6-Fluc site even at 10 min after injection of both substrates. There is a clear distinction in the pattern of light kinetics with each reporter maintaining its individual characteristics. The control site shows background level of signal. The results are consistent across three different mice. Discussion:

The present inventor has shown that Rluc can be used in living mice by measuring light from RL bioluminescence in a cooled CCD camera after mice are tail vein or i.p. injected with coelenterazine. The present inventor has shown that Rluc can be transiently transfected into a variety of cell types and that the kinetics of light production from cell culture lysates are rapid, with a quick peak in the initial 10 seconds followed by a rapid decline over 10 minutes. The present inventor has further shown that the peak signal can be increased with increasing coelenterazine dose up to a limit, followed by a decrease in signal. This decrease was unexpected but may be due to color changes in the buffer, which probably lead to greater absorption of bioluminescent light. There was no significant bioluminescence when cells in culture transiently expressing Rluc were exposed to D-luciferin or when cells transiently expressing Flue were exposed to coelenterazine. These in vitro and cell culture data support the unique characteristics of Rluc and also provide a basis for using both Rluc and Flue in the same living animal, due to the lack of significant crossreactivity of RL and FL for their respective substrates.

The present inventor has extended these results to living mice, demonstrating the ability to image C6-Rluc cells implanted in the liver, lungs, peritoneum, and s.c. regions of living animals. The ability to image cells in deeper tissue was achieved by injecting cells via the tail vein and letting them naturally traffic to the lungs via the liver. The present inventor has shown that higher doses of coelenterazine injected via tail vein can be used to detect C6- Rluc cells from deeper tissues as compared with superficial tissues. This result is likely due to less transmitted light from deeper tissues, and formal quantitative relationships between depth and light transmission will help to better characterize this. Although most of the bioluminescent light is likely to be scattered and absorbed, enough escapes from the animal to be detected by the highly sensitive cooled CCD camera.

The present results show that bioluminescence can also be produced while injecting coelenterazine i.p., although the kinetics of light production from nonperitoneal sites are slower. This observation is probably due to a slower transit time for coelenterazine to get into the blood from the peritoneal space.

The kinetics of light production from regions containing C6-Rluc cells depends on the route of coelenterazine injection as well as on the specific site of the cells. Increasing the dose of tail vein injected coelenterazine in mice s.c. implanted with C6-Rluc cells shows an increase in the CCD measured light. Studies to compare the sensitivity in terms of photon yield/concentration of bioluminescent protein/concentration of substrate will further elucidate the differences between Flue and Rluc. It is also important to note that C6 control cells implanted in mice show only background signal when mice were tail vein or i.p. injected with coelenterazine. Control mice with no cells implanted and injected with coelenterazine also only show background signal. The background signal is due to various factors including low levels of light emitted from the mice even though there is no bioluminescent light, low levels of photons in the "light tight" box, as well as noise from the CCD camera due in part to thermal drift.

Results from mice carrying both the C6-Rluc and C6-Fluc cells implanted s.c. show that the RL and FL signal can be distinguished by separate injections of D-luciferin and coelenterazine, as was also seen in cell culture studies. Although the FL:D-luciferin signal was much higher, it must be noted that the amount of coelenterazine used was relatively low compared with D-luciferin to minimize costs. D-luciferin was purchased at a cost of $5/mg and coelenterazine at a cost of $190/mg. However, the effective cost per mouse study for D- luciferin was $15 (3 mg/mouse), and $19 for the highest dose of coelenterazine used (100 μg/mouse). With increasing use of coelenterazine, it is likely that improved methods for its synthesis and demand will further reduce costs.

In mice carrying both C6-Rluc and C6-Fluc cells implanted s.c, which were injected simultaneously with D-luciferin and coelenterazine via tail vein, distinct light kinetics from RL and FL were observed. Light from the C6-Rluc cells quickly peaked and was rapidly extinguished, whereas light from C6-Fluc cells peaked later and persisted longer. The difference in light kinetics should allow separation of reporter protein signal through obtaining multiple images in the same mouse after co injection with both D-luciferin and coelenterazine.

Several issues could have hindered the success of imaging Rluc expression with coelenterazine in living mice. These issues include instability of coelenterazine in plasma, insufficient delivery of coelenterazine to target sites, as well as insufficient bioluminescent light yield.

The surprising success of using Rluc in living organisms should yield some distinct advantages over Flue. RL is a 36 kDa monomeric enzyme that catalyzes the oxidation of coelenterazine in presence of oxygen to generate a flash of blue luminescence with a wavelength centering at 482 nm. The oxidative decarboxylation of coelenterazine by RL in the presence of oxygen yields "oxyluciferin" CO₂ and blue light (λ_max = 480 nm) in vitro (Wilson, T. & Hastings, J. W. (1998) Annu. Rev. Cell Dev. Biol. 14, 197-230). By contrast, FL is a 61 kDa single subunit protein that catalyzes D-luciferin to produce oxyluciferin in the presence of oxygen, cofactors, Mg⁺², and ATP to give a flash of green light at 562 nm (DeLuca, M. & McElroy, W. D. (1974) Biochemistry 13, 921-925.). It should be noted that the two proteins RL and FL and the two substrates coelenterazine and D-luciferin are structurally unrelated. The RL coelenterazine reaction is relatively simple compared to the FL D-luciferin reaction (Liouye, S. & Shimomura, O. (1997) Biochem. Biophys. Res. Commun. 233, 349- 353). RL has a significant advantage in that it does not need cofactors or ATP, and therefore Rluc is ideal because it likely causes less perturbation to the cells in which it is expressed. Also, the rapid light kinetics of RL in living mice is likely to be quite useful in animal experiments where a quick signal is needed that does not persist over time. Future studies directed to the half life of Rluc mRNA and RL should yield insight into the ability to repetitively image changes in reporter gene expression. Furthermore, quantitative relationships between levels of Rluc mRNA, RL, coelenterazine delivery, and bioluminescence will also be useful for building fully quantitative assays.

The current study has not addressed the biodistribution of coelenterazine in mice. Previous studies with Flue and D-luciferin have also not addressed the biodistribution of D- luciferin but have found through placing cells in various sites, using various gene delivery vectors, and transgenic models the high accessibility of D-luciferin to various tissues, including the brain. It is anticipated that coelenterazine will also be accessible to many tissues because of its diffusable nature (Lorenz, W. W., et al. (1996) J. Biolumin. Chemilumin. 11, 31-37).

Such biodistribution studies may be pursued by radiolabeling both coelenterazine and D-luciferin , then tracking their distribution throughout the body of a living organism. It will be important to perform these studies with different doses of cold and radiolabeled substrate to study any changes in biodistribution. Furthermore, potential differences in biodistribution between mice and rats may be studied.

The current study has not addressed any potential toxicities of repetitively using coelenterazine in living mice, although no direct toxicity was observed in these mice studies. Formal toxicology studies may be performed as is known in the art. The stable expression of Rluc gene in C5 fibroblast has indirectly demonstrated the nontoxicity of the Rluc gene product in mammalian cells, as reported (Lorenz, supra). Toxicology studies have also not yet been reported for D-luciferin.

As may be appreciated by one of skill in the art, these studies demonstrate the feasibility of using Rluc in various paradigms in living subjects, including gene delivery, cell trafficking, and transgenic models. For example, Rluc may be used with Flue in numerous models in which two independent optical signals are desired for measurement of the location, magnitude, and persistence of expression of two different genes.

It is anticipated that mutants of Rluc may be identified or produced that have significant wavelength differences from the wild type, and synthetic versions of Rluc may be better optimized for efficient expression in mammalian cells. RL in combination with FL and its mutants will allow multiplexing approaches in which several molecular events can be simultaneously studied through the use of multiple reporter genes, each with proteins that have distinct signals due to wavelength, substrate specificity, or both.

Example 2 Optical Imaging of Renilla luciferase, Synthetic Renilla Luciferase, and Firefly Luciferase Reporter Gene Expression in Living Mice

As shown above, Renilla luciferase (Rluc) is a bioluminescence reporter gene that can be used for non-invasive optical imaging of reporter gene expression in living mice, with the aid of a cooled Charged Couple Device (CCD) camera. In this example, a synthetic Renilla luciferase reporter gene (hRluc) from Promega (B. B. Zhuang Y, Hawkins E, Paguio A, Orr L, Wood MG, Wood KV, Promega Notes 79, 6-11 (2001)) is utilized, wherein the native Rluc gene has been redesigned and the codon usage optimized to improve the expression of the reporter. The number of transcription factor binding sites has been reduced from 300 in the Rluc to 4-5 sites in hRluc. In adddition, deletion of poly (A) additional signals (AATAAA) and incorporation of a kozak sequence at the beginning of the gene has been used for better expression efficiency. (M. Kozak, J Mol Biol 196, 947-950 (1987)). The resulting reporter gene is expected to have higher transcriptional efficiency, which should greatly enhance the detection of the reporter enzyme in vivo. In this example, gene expression of hRluc is compared to that of Rluc both in cell culture and in living mice. To date, Flue has been more frequently used as a bioluminescence reporter gene for non invasive, real time imaging studies. The present inventor therefore also compared the signal differences of hRluc and Flue for real time noninvasive imaging from living mice. Materials and Methods:

Cell Lines, Culture Conditions, and Transfection Procedures. C6 rat glioma cells were maintained in glucose deficient Minimum Eagle's Medium (MEM) supplemented with 1% penicillin-streptomycin, 1% L-glutamine and 5% Fetal Calf Serum (FCS). N2a (mouse neuroblastoma) cells, COS-1 (green monkey kidney) cells and 293T (human kidney) cells were maintained in DMEM supplemented with 1% Penicillin-Streptomycin and 10% FCS.

For assessment of RluclhRluc expression in various cell types, each cell type described above was plated in 12- well plates (2 x 10⁵ cells/well) and transfected with pCMV- Rluc/pCMV-hRluc plasmids (pRL-CMV, phRL-CMV, Promega, Madison, CA) and co- transfected with pCMN β-gal, using SuperFect Transfection Reagent (Qiagen, Valencia, CA) to normalize for transfection efficiency. Mock-transfected cells were used as control. Cells were lysed in lysis buffer 24 hr post transfection, and biochemical studies were carried out using a luminometer as described below. For comparing Flue expression, C6 cells were transfected with pCMV-FZwc plasmid (provided by CH. Contag, Stanford University, Stanford, CA) and co-transfected with pCMV β-gal in parallel 12-well plates. The transiently transfected cells are referred to as C6-Rluc, C6-hRluc and C6-Fluc in the following studies.

C6 and 293T cells were also grown in 35 mm and 100 mm plates (Costar), and transfected with pCMV-RZwc or pCMV -hRluc under similar conditions. They were collected by trypsinization 24 hr post transfection, washed with PBS, counted, and various concentrations of cells, resuspended in PBS, were implanted in mice for in vivo studies as described below.

Preparation of coelenterazine and D-Luciferin. Coelenterazine (also known as "native coelenterazine"), a substrate for RL/hRL, was purchased from Prolume Ltd. (Pinetop, AZ). The compound (2 mg/ml) was dissolved in absolute ethanol. Further dilutions were made in 50 mM sodium phosphate buffer (SPB), pH 7. D-Luciferin firefly potassium salt, the substrate for FL was purchased from Xenogen Corp. (Alameda, CA). A 30 mg/ml stock in PBS was prepared and filtered through 0.22 micron filters before use.

Luminometer Measurements. All bioluminescence assays were performed in a TD 20/20 luminometer (Turner Designs, Sunnyvale, CA). Five microliters of crude and clarified cell lysates obtained from C6-Rluc, C6-hRluc and mock-transfected C6 control cells were mixed with 100 μl of coelenterazine, prepared at various concentrations of 0.0001,0.001,0.01,0.1, 1.0, 10, 50 and 100 μg/ml in SPB. The dose dependent relative light units (RLU) were recorded in the luminometer for 10 seconds. The lysates were collected from three separate wells for each dose and the bioluminescence was normalized to total cellular protein of the well. The protein content of the cell lysates were mixed with Bio-Rad protein assay reagent (Bio-Rad Laboratories, USA) and recorded in a Beckman DU-50 spectrophotometer (Beckman Instruments Inc., USA). The luminescence results were reported as RLU per microgram of protein/second. The results are normalized to β-gal activity and represented as RLU/μg/sec/β-gal activity. Cell lysates (5 μl) from C6-Fluc cells were mixed with 100 μl of LARII (Promega). The RLU was recorded in the luminometer and normalized to total cellular protein. The results are reported as RLU/μg/sec/βgal activity.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR), SDS-PAGE and Western blotting. RT-PCR was conducted to compare the level of messenger RNA produced by C6 cells transiently transfected with pCMV-RZwc and pCMV-hRluc and mock transfected C6 control cells. Equal number of cells (2.5 x 10⁶) were plated to carry out the transfection experiments. RNA extraction was done from transfected C6 and control cells using RNeasy kit (Qiagen). The RNA was first treated with RQ1 Rnase-Free Dnase (Promega) to degrade any contaminated double and single stranded DNA prior to RT-PCR. Removal of DNA was confirmed by doing PCR with the treated RNA. The GAPDH housekeeping gene was used as a positive control and levels of expression were near equivalent in the four cell lines studied. The RT-PCR was done with the aid of GeneAmp EzrTth RNA PCR kit (Applied Biosystems) using separate primers designed against Rluc and hRluc. Rluc primers had the region flanking 64 - 83 bp as sense and region flanking 267-247 bp as antisense whereas hRluc primers had the region flanking 162-181bp as sense and the region flanking 364-345 bp as antisense. A 50 μl of reaction contained 200 ng of total RNA, 100 ng each of RluclhRluc forward primers and RluclhRluc reverse primers, IX reverse transcription buffer (100 mM Tris-HCl, pH 8.3, 900 mM KCl with 1 mM MnCl₂), 200 μM dNTPs and 10 units of vTth DNA polymerase. RT reactions were performed at 60°C for 45 minutes followed by 1 min pre-heating at 94°C and 35 cycles of PCR amplification at 94°C for 1 min, annealing and extension at 48°C for 1 min followed by a final extension at 72°C for 15 min. The reaction products (lOμl) were separated in a 2% agarose gel stained with ethidium bromide. The band intensity was further quantified with Kodak ID. 3.5 image analysis software and the Mean Intensity recorded.

RT-PCR was also conducted to compare the level of messenger RNA produced by C6 cells transfected with pCMV-hRluc and pCMV-Fluc and C6 control cells. RNA extraction was done from transfected C6 and control cells and further treated with RQ1 Rnase-Free Dnase (Promega) to degrade any contaminated double and single stranded DNA prior to RT- PCR. The RT-PCR was done with the aid of GeneAmp EzrTth RNA PCR kit (Applied Biosystems) using primers designed against Flue. The hRluc primers were same as above, whereas Flue primers had the region flanking 92-111 bp as sense and region flanking 293- 274 bp as antisense. The 50 μl of reaction contained 200 ng of total RNA, 100 ng each of FluclliRluc forward primers and reverse primers, IX reverse transcription buffer, 200 μM dNTPs and 10 units of rTth DNA polymerase. Equal volume of reaction products (lOμl) were separated in a 2% agarose gel stained with ethidium bromide. The band intensity was further quantified with Kodak ID. 3.5 image analysis software and the mean intensity recorded.

For Western blot, protein extracts were prepared from C6 cells transiently transfected with pCMV-RZwc and pCMV-hRluc. Mock transfected C6 cells were used as a control. Cells (1 x 10⁶) were washed and boiled in Laemmli's sample buffer. Equal amount of protein (10 μg) was resolved on 10% SDS-PAGE and transfered to nitrocellulose membrane (Immun- Blot PVDF Membrane, Bio-Rad). Immunoblotting was performed using anti-Renilla antibody (1:1000 μl, Chemicon Int.). The blot was washed and incubated with anti-mouse IgG coupled to Alkaline Phosphatase (1:3000 μl, Chemicon Int.) and developed with BCJT/NBT (Roche Diagnostics).

Imaging and Quantification of Bioluminescence Data. The in vivo Imaging System (IVIS™) (Xenogen Corp., Alameda, CA) consisting of a cooled CCD camera mounted on a light-tight specimen chamber (dark box), a camera controller, a camera cooling system, and a Windows computer system for data acquisition and analysis was utilized. (J. C. Wu, G. Sunderasan, M. Iyer and S. Gambhir, Molecular Ηierapy A, 297-306 (2001)). Each supine/prone mouse was placed in the specimen chamber mounted with the CCD camera cooled to -120°C, with a field of view FOV set at 25 cm height above the sample shelf. The photon emission, transmitted from mice was measured. The gray scale photographic images and bioluminescence color images were superimposed using the Livinglmage v. 2.11 software overlay (Xenogen Corp., Alameda, CA) and Igor image analysis software v. 4.05 (Wavemetrics, Lake Oswego, OR). A region of interest (ROI) was manually selected over the signal intensity. The area of the ROI was kept constant and the intensity was recorded as maximum (photons/second/cm²/steredian (sr)) within an ROI.

Coelenterazine Dose Studies. All animal handling was performed in accordance with University of California, Los Angeles (UCLA) Animal Research Committee guidelines. Five sets (N=2) of CD-I mice (~ 30 g; Charles River Breeding Laboratories), 4 weeks old, were anesthetized by intraperitoneal injection of - 40 μl of a ketamine and xylazine (4:1) solution, followed by subcutaneous implantation of C6-hRluc cells (1 x 10⁶ cells in 100 μl PBS) in the left forearm region and C6 control cells (1 x 10⁶) in the right thigh region. Different doses of coelenterazine (0.07, 0.36, 0.7, 1.4, 2.1 mg/kg body weight) were injected via tail-vein in duplicate mice. Bioluminescence was measured from both C6 control and C6-hRluc sites, over a 5 minutes time period using 10- thirty second acquisition scans.

Simultaneous Imaging of RL and hRL Bioluminescence in Mice. Two sets of CD- 1 mice, were anesthetized as described above. To check for background signal from animals not expressing the Renilla luciferase reporter gene, one set of mice (N=2) was tail-vein injected with 0.7mg/kg body weight of coelenterazine. Another set of mice (N=3) were injected at 3 subcutaneous sites with C6-Rluc (1 x 10 cells) in the left forearm, C6-hRluc (1 x 10 cells) in the right forearm and C6 control cells (1 x 10 ) at the right thigh region. One hundred μl of coelenterazine (0.7 mg/kg body weight) was injected via tail- vein and a whole- body image of the mice were acquired with 15 one-minute scans using the cooled CCD camera. Equal number (2 x 10⁶) of different batches of cells (C6, C6-Rluc, C6-hRluc) were lysed with lysis buffer and the signal recorded in the luminometer prior to implantation into CDl mouse.

Sensitivity Studies. Experiments were conducted on nude mice to minimize background signals from the body fur. Mice were anesthetized by i.p. injection of - 10 μl of a ketamine and xylazine (4:1) solution. 293T cells, transiently transfected with pCMY-hRluc plasmid (referred to as 293T-hRluc) were collected 24 h post transfection. Separate groups of nude mice (N=2) were implanted with different number of cells (10 and 100 cells) at subcutaneous sites using a 5 μl hamilton syringe. Tail- vein injection of coelenterazine (1.4 mg/kg body weight) was performed. The images were acquired for 5 minutes under the cooled CCD camera.

Simultaneous Imaging of FL/hRL Bioluminescence from Subcutaneous Sites in Living Mice. CDl mice (N=3) were anesthetized and implanted (s.c) with C6-Fluc (1 x 10⁶ cells) in the right forearm, C6-hRluc (1 x 10⁶ cells) in the left forearm and C6 control cells (1 x 10⁶) at the right thigh region. A mixture of D-Luciferin (150 mg/kg body): coelenterazine (0.7 mg/kg body) was injected via tail-vein and whole-body images of the mice were acquired with 15 one-minute scans using the cooled CCD camera.

Imaging of FL hRL Bioluminescence in Living Mice from Deep Tissues. CDl mice (N=3), 4 months old (-35 g each), were injected with a mixture of C6-Fluc cells: C6- hRluc cells = 1:1 (2 x 10⁶ cells total) via tail vein. The cells traveled via the circulation and traffic into lungs. Two hours later, the mice were anesthetized followed by intravenous injections of high doses of coelenterazine (5.7 mg/kg body weight). A whole-body image of the mice were acquired with 3 one-minute scans using the cooled CCD camera. Two hours later, the mice were re-scanned to check for the residual signals from the implanted C6-hRluc cells. When no signal was detected, the same set of mice were injected with D-Luciferin (150 mg/kg body) given intraperitoneally followed by the whole-body images of the mice acquired with 3 one-minute scans using the cooled CCD camera. Results: pCMY-hRluc transfected cells show significantly higher signals as compared to pCMVR wc transfected cells across different cell lines tested. Cell lines from different tissue origins (C6, N2a, 293T, COS-1) were transiently transfected with either pCMV-RZwc or pCMV-hRluc plasmids. The difference in the expression of the two Renilla luciferase reporter genes was compared. All cell lines shows significantly higher (P<0.05) levels of gene expression from ipCMV-hRluc transfected cells when assessed by the luminometer (FIG. 8). However the level of expression varied in different cell lines with 293T showing -500- fold, COS1 -800-fold, N2a -1500-fold and C6 -150 fold higher expression from pCMV- hRluc transfected cells as compared to pCMV-RZwc transfected cells. Successful transfection in different cell lines indicates that hRluc has consistent high expression level in different tissues.

Increasing signal is observed from C6-hRluc cell lysates with increasing doses of coelenterazine. To compare the light yield from C6-Rluc and C6-hRluc cell lysates with increasing doses of coelenterazine, different doses (0.0001-100 μg/ml) were prepared in 50 mM SPB by serial dilution. Coelenterazine as low as 0.0001 μg/ml is able to produce a measurable light signal in the luminometer from C6-hRluc cell lysates but not with C6-Rluc or C6 control cell lysates. A maximum signal of 1.0 x 10⁶ ± 4.2 x 10 RLU/μg/sec/β-gal activity from C6-hRluc cell lysates and 2.8 x 10³ ± .72 x 10² RLU/μg/sec/β-gal activity from C6-Rluc cell lysates were recorded in the luminometer at lμg/ml dose (FIG. 9). There is an -250-fold higher signal with hRL as compared to RL at every coelenterazine dose. An approximately linear relationship (hRL; R²=0.98, RL; R²= 0.96) between the doses of coelenterazine (0.01 - 1 μg/ml) and signal intensity is observed from both samples. At higher doses (>1 μg/ml) there is a plateau and eventually a drop in light production. RT-PCR and Western Blot analysis shows higher message level and protein production from C6-hRluc cells as compared to C6-Rluc cells. In order to compare the transcription and translation levels in the C6-Rluc and C6-hRluc cells, RT-PCR and western blot was performed (FIG 10). The RT-PCR study (FIG. 10a) shows a higher level of message production from C6-hRluc cells seen as a stronger band (lane 2) as compared to C6- Rluc cells (lane 3). Both amplified a product size of 203 bp. The control cells do not show any detectable band in the blot (lane 4). The mean intensity (MI) of the hRluc band is 117.16 and Rluc band is 90.23 whereas control area is -65.52.

There is also a corresponding higher production of hRL vs RL as seen in the western blot (Fig. 10b). C6-hRluc cell lysates show a more intense band (lane 2) than do C6-Rluc cells lysates (lane 3). The control (lane 1) shows no detectable signal in the blot. hRL signal enhances with increasing coelenterazine dose in living mice. C6- hRluc cells were subcutaneously implanted into the left forearm of mice (N=2) while mock- transfected C6 cells were implanted in the right thigh. Mice injected with very low doses of coelenterazine (0.07 mg/kg body weight) via tail-vein show measurable signal of - 1.6 x 10⁵ ± 1.1 x 10⁴ maximum (p/s/cm²/sr) from a ROI drawn on images over the site of implantation at the left shoulder area. There is a progressive increase in the bioluminescence from the implanted cells with increasing coelenterazine dose from 0.07 - 2.1 mg/kg body weight (FIG. 11). The C6 control site at the right thigh region shows a signal of - 5.8 x 10³ ± 8.0 x 10² - 1.3 x 10⁴ ± 1.9 x 10³ maximum (p/s/cm²/sr).

C6-hRluc cells yields significantly higher signal than C6-Rluc cells when both set of cells are implanted in the same living mouse and imaged simultaneously. To compare the signal emission from a living mouse by a cooled CCD camera, C6-Rluc, C6-hRluc and C6 control cells were implanted at three separate s.c. sites in living CD-I mice (N=3). Coelenterazine (0.7 mg/kg body weight) was injected via tail vein. There is - 25-30 fold difference in the detectable signal by cooled CCD camera between the two reporters, with synthetic Renilla showing significantly higher (P<0.05) and prolonged signal emission (FIG. 12a). The C6-hRluc site shows signals up to 4.3 x 10⁶ ± 2.4 xlO⁵ maximum (p/s/cm²/sr) in the first minute of scan which drops to 4.1 x 10⁵ ± 3.9 x 10⁴ maximum (p/s/cm²/sr) after 15 minutes whereas the signal from the C6-Rluc implanted site is 1.5 x 10⁶ ± 0.8 x 10⁴ maximum (p/s/cm²/sr) and drops to 1.7 x 10⁴ ± 0.72 x 10³ maximum (p/s/cm²/sr) within 15 minutes (Fig. 12b). Control mice with no implanted cells, when injected with coelenterazine (0.7mg/kg body weight), show a background signal of - 3.1 x 10³ ± 0.5 x 10² maximum (p/s/cm²/sr).

The lysates from the same batches of C6-hRluc cell shows 104 fold higher signals (4.9E+03±5.1E+02 RLU/_g/sec) compared to C6-Rluc cell lysate (4.7E+02±5.1 RLU/_g/sec) when checked in the luminometer. C6 control cell lysates had negligible signal. Measurable signal is detected from living mice subcutaneously implanted with 10-100 293ThRluc cells. Since 293T-hRluc cells were observed to give the highest signal in the luminometer (Fig. 8), these cells were subcutaneously implanted in nude mice (N=2) to check for the minimum number of imagable cells. Detectable signals (P<0.05) were seen from as few as 10 cells (2.25 x 10⁴ ± 7.3 x 10³ maximum (p/s/cm²/sr)) and 100 cells (4.4 x 10⁴ ± 2.9 x 10⁴ maximum (p/s/cm²/sr)) implanted at s.c. sites by hamilton syringe in low volume (5 μl) of buffer. The nontransfected control cells show a background signal ~2.5xl0³ ± 2.3xl0² maximum (p/s/cm²/sr).

RT-PCR and luminometer studies show a higher message level and signal production from C6-hRluc cells as compared to C6-Fluc cells. The RT-PCR study (FIG. 13 a) shows a higher level of message production from C6-hRluc cells seen as a stronger band (lane 4) as compared to C6-Fluc cells (lane 2). Both amplified a product size of -203 bp. The control cells do not show any detectable band in the blot (lane 1). The mean intensity (MI) of the hRluc band is 117.16 and the MI of the Flue band is 91.12, whereas the control area is - 65.52.

In cell culture experiments, C6 cells transfected with pCMV-FZwc shows -17-20 fold lower signal in a luminometer as compared to C6-hRluc cell lysates when normalized to the β-Gal activity (Fig 13b).

C6-hRluc cells yield significantly higher signal than C6-Fluc cells when implanted subcutaneously in the same living mouse. C6-Fluc and C6-hRluc cells were implanted at two separate s.c. sites in the same living CD-I mice (N=3) and a mixture of D- Luciferin : coelenterazine was injected via tail vein in order to compare the signal emission from two different reporters using two different substrates. The signal from C6-hRluc cells (left forearm) peaks up to 4.7 x 10⁶ ± 6.7 x 10⁴ maximum (p/s/cm²/sr) within the first minute after coelenterazine injection. However, C6-Fluc cells implanted on the right forearm take -8-10 minutes to reach the peak signal (1.3 x 10⁵ ± 1.7 x 10⁴ maximum (p/s/cm²/sr)). A detectable signal from the C6-Fluc implanted site can be first seen - 3-5 minutes after the injection of the substrate by the cooled CCD camera (data not shown). The measurable signals from the C6-hRluc cell implanted site were - 30 - 40 fold higher than C6-Fluc cells. C6 control cells implanted at right thigh region show only background signal (FIG. 14).

C6-hRluc yields higher signals than C6-Fluc cells when cells traffic to lungs in the same living mouse. C6-Fluc and C6-hRluc cells were mixed and injected via tail vein in the same mice so that the cells could traffic to deeper lung tissue. The substrates coelenterazine and D-Luciferin were injected separately in order to compare the signal emission from the two different reporters from the same site. The signals from both the reporters were emitted from the lung region as seen in FIG. 15 a. The signal from C6-hRluc cells peaks up to 1.2 x 10⁵ ± 4.4 x 10⁴ maximum (p/s/cm²/sr) within the first minute after coelenterazine injection (FIG. 15.a.l), whereas C6-Fluc cells took around -8-10 minutes to reach the peak signal of 2.8 x 10⁴ ± 2.7 x 10³ maximum (p/s/cm²/sr) by the cooled CCD camera (FIG. 15.a.2). It should be noted that a higher dose of coelenterazine (5.7 mg/kg body weight) was used for these experiments. We also selected larger adult mice with maximum body weight to study the signal emission from deep tissues. The measurable signals from the C6-hRluc cells in the lungs were - 4 fold higher than C6-Fluc cells even from the deeper tissues (FIG. 15b). Discussion:

In Example 1, it was shown that Rluc can be successfully used for real time imaging studies in living animals using a cooled CCD camera. Upon tail- vein injection of coelenterazine, the RL enzyme in the presence of coelenterazine and oxygen generates a flash of blue luminescence with a wavelength centered at - 482 nm. In Example 2, an improved version of the Renilla luciferase reporter gene, termed synthetic Renilla luciferase (hRluc), was used. Though the nucleotide sequence of hRluc retains only 72% homology with the native Rluc, the amino acid sequence is unchanged (36 kDa protein) producing the same light yield centered at - 482 nm.

It was initially demonstrated that the hRluc reporter gene is well adapted for mammalian cells and can be transiently transfected into a variety of cell types. In all the cell lines tested, there is a significant increase in the measured signal with hRluc as compared to Rluc while taking into account transfection efficency. The absolute amount varied with cell type as shown in Fig. 8. The kinetics of light production from C6-hRluc cell culture lysates is rapid and -100 - 250 fold higher than C6-Rluc cell lysates when studied with various doses of coelenterazine. The signal from hRluc cell lysates peaks with increasing coelenterazine dose and then decreases similar to the signals from Rluc. The higher yield of signal from pCMV- hRluc transfected cells is due to a significantly higher level of transcription efficiency leading to more mRNA production as verified by RT-PCR (Fig. 10a). Subsequent up-regulation of luciferase enzyme (hRL) in cells is further confirmed by western blot analysis using an anti- Renilla antibody (Fig. 10b).

The results in living mice carrying both C6-Rluc and C6-hRluc cells implanted subcutaneously, and tail-vein injected with coelenterazine, show differences in light yield from the two sites of cell implantation and correlated to the cell culture data. The typical pattern of flash kinetics was observed within the first minute of coelenterazine injection from both sites. The magnitude of the signal from the C6-hRluc cell implanted site is about - 25- 30 fold more than that of C6-Rluc cells. The relative advantage in vivo is still quite high as expected, but the absolute fold drops primarily due to light attenuation in tissue. The light from the C6-Rluc cells rapidly extinguishes within 15 minutes, whereas the signal from C6- hRluc cell implanted site tends to persist beyond 15 minutes. As with C6-Rluc cells, the kinetics of measurable light production from regions containing C6-hRluc cells is dependent on the dose of coelenterazine injection. However with C6-hRluc, even a very low dose of coelenterazine (0.07 mg/kg body weight) yields a measurable signal with the cooled CCD camera. C6 control cells implanted in mice shows only background signal when mice were tail vein injected with coelenterazine. The background signal of -3.1 x 10³ ± 0.5 x 10³ maximum (p/s/cm /sr) from control mice with no implanted cells, could be due to various factors including low levels of light emitted from the mice even though there is no bioluminescence, low levels of photons in the "light-tight" box, as well as noise from the cooled CCD camera due in part to thermal drift.

Since cells transfected with synthetic Renilla luciferase show an improvement in signal level, it is important to study the minimum number of cells that lead to detectable signal. 293T-hRluc cells were selected for this purpose, as they show maximum signal in the cell culture study (Fig. 8). Results show measurable signal from as low as 10 cells when implanted at a subcutaneous site of a living mouse. It should be noted that the 10 cells were implanted in 5 μl PBS using a 5 μl hamilton syringe, so that the cells were at close proximity. Injecting very few cells in higher volume of buffer gave inconsistent results, as the cells were spread over a larger area. However 100 cells at a subcutaneous site gave a significant and consistent signal. Such observations may aid in future cell trafficking studies involving fewer cells. (S. Mandl, et al, I.Cell Biochem Suppl 39, 239-248 (2002).) This improvement in the signal yield from C6-hRluc cells from small animals can be useful for several experimental strategies including: (i) significant signal with lower doses of substrate injection to animals (ii) detectable signal from much fewer number of cells or (iii) gene expression involving rare molecular events/weak promoters could be more easily imaged.

To date, Flue is the most thoroughly characterized reporter gene used for noninvasive imaging studies in small animals with optical bioluminescence approaches. (V. Ntziachristos, et al, Nat. Med. 8, 757-761 (2002).) In the present study, the signal of FL was compared with that of hRL both in cell culture and from mice to see which is more sensitive for noninvasive studies. No absolute comparison of the two reporters was performed as there are differences in gene size, reaction parameters, substrate chemistry, as well as substrate pharmacokinetics. The lack of significant cross reactivity between the two reporters and their corresponding substrates provides for a basis to study and compare both hRluc and Flue in the same living mouse. FL: D-Luciferin produces light with a peak in the range of 595-620 nm in presence of oxygen, ATP and the cofactor magnesium whereas hRL: coelenterazine produces light which peaks at - 482nm. Since the optical properties of mammalian tissue involves attenuation of light <600 nm, the chances of acquiring measurable signals from Flue is greater. However the cell culture study shows - 120-fold more signal in the luminometer from C6-hRluc than those 0,6-Fluc cell lysates. Also, the measurable signal from C6-hRluc is found to be - 30-40 fold more than C6-Fluc when cells are implanted subcutaneously in the same mouse and imaged by the cooled CCD camera. There can be several reasons for lower detectable signal from C6-Fluc cells. The FL:Dluciferin reaction requires ATP from the cells to generate the light. This could be a limiting factor for higher signal yield as the reaction may subside with lower levels of cellular ATP. Also, utilizing cellular ATP reserve for the reaction may perturb cellular machinery. One key advantage of the

RZwc//-RZz-c:coelenterazine reaction is that it does not utilize any ATP. It is known that the signal wavelength of hRluc: coelenterazine is centered at - 482 nm where chances of attenuation of light from mammalian tissue is greater. There are also reports of faster rate of coelenterazine degradation and inactivation in tissues. (C. E. O'Connell-Rodwell, S. M. Burns, M. H. Bachmann and C. H. Contag, Trends in Biotechnology: A TRENDS guide to Imaging Technologies 20, S19-S23 (2002)). However the initial light yield from the hRL: coelenterazine reaction is so high that the measurable light is significantly higher than that of Flue reporter even after attenuation by tissue at the depths and locations tested. It should be noted that the signal from the mouse could be controlled by the amount of substrate injected in each animal. There is a significant difference in the substrate dose of coelenterazine and D-Luciferin injected in each group of mice. The dose of D-Luciferin is -150 mg/kg body weight and that of coelenterazine is only - 0.7 mg/kg body weight for most of the studies. With even a 150-fold lower dose of substrate (coelenterazine), -30-40 fold more signal is seen from C6-hRluc implanted cells as compared to Q.6-Fluc cells from same the mouse at the s.c. site. However, there is a marked decrease in the signal difference when signals are acquired from C6-hRluc cells present in deeper tissues such as the lungs. This was compensated for, by injecting higher doses of coelenterazine (-5.7 mg/kg) via the tail-vein. For the higher dose of coelenterazine, the signals emitted from C6-hRluc cells in lungs were - 4 fold higher than C6-Fluc cells situated in the same location. It should be noted that since equal numbers of cells are used, and there is a difference in transfection efficiency of -30% in favor of ipCMV-hRluc, that a more accurate fold-difference is -4 x 0.7 = -3-fold at the particular doses of coelenterazine and D-Luciferin utilized. However, in order to do an absolute comparison between Renilla and firefly luciferase, one would need to know the exact levels of active FL and hRL, as the levels of transcription are likely different, even though transfection efficiency can be corrected for. The exact dose of D-Luciferin and coelenterazine are two additional variables that will need to be optimized for a given experimental strategy. It will be important in future studies to also study other deep tissue sites of expression (e.g., liver, brain, etc.). It is also possible that D-Luciferin and/or coelenterazine efflux from cells could be affected by levels of P-glycoprotein and other multi-drugresistance mechanisms, and this will also have to be further investigated. Three repeated doses of coelenterazine as high as -3.5 mg/kg body weight and a single dose as high as 5.7 mg/kg did not show any toxicity effects of sudden death, lethargy, weight loss, and changes in vital signs. Formal toxicology studies will be needed to better define any potential toxicity of coelenterazine as a function of dose and frequency of administration. Formal toxicology studies for D-Luciferin are also needed. It should also be noted that the current costs for coelenterazine and D-Luciferin are approximately $75/mg (Prolume Ltd.) and $0.80/mg (Xenogen Corp.) respectively. Therefore at the typical doses used for each substrate (micrograms of coelenterazine and milligrams of D-Luciferin), the costs per mouse study are near equivalent. Additional work to further characterize relative advantages/disadvantages of Flue and hRluc in living subjects should help to further accelerate bioluminescence based research.

The use of bioluminescence in various paradigms in living subjects including gene delivery, cell trafficking and transgenic models is already possible. The present inventor has now shown that hRluc is a more sensitive primary reporter gene as compared to Rluc in various in vivo studies. The present inventor has also shown that the hRluc signal is significantly higher than Flue at subcutaneous sites under transient transfection conditions. For lung imaging, higher doses of coelenterazine were needed in order to achieve signal greater than that with Flue. The current work helps further clarify the issues surrounding the use of Rluc, hRluc, and Flue as reporter genes for bioluminescence imaging in living subjects.

Other Applications of Renilla luciferase Based Assay in Living Organisms. Studying intact organisms allows for preserving the normal environment in which cells are found, thereby preserving their connections and feedbacks to all other cells in the organism. Future studies of integrated organisms will likely rely on techniques to study the intact organism with minimal perturbation.

The present inventor has shown herein that the genes Rluc and hRluc, which can be used in such in vivo studies as a marker for cell migration, gene expression, study of endogenous gene expression, protein interaction, etc., can be implanted into a living animal, and that coelenterazine, when injected into the living animal, sufficiently disperses throughout the organism to act as a substrate for RL or hRL being expressed in the animal.

The present inventor has further shown that the resulting bioluminescence in the living animal can be detected, recorded and quantified. Thus, for example, cells containing Rluc and hRluc can be injected into a living animal and the subsequent movement of the cells within the body monitored by the administration of coelenterazine, followed by the detection and recordation of bioluminescent light.

Similarly, gene expression can be measured in vivo by linking a promoter of interest to Rluc or hRluc, administering coelenterazine and measuring the resulting bioluminescence in response to various factors affecting gene expression. Also, since signals were measured from deeper tissues (e.g., lungs), it is likely that transgenic mice could be created carrying the rl or hrl reporter genes linked to a promoter of choice, allowing measurement of various biological processes in the context of an intact animal.

The present inventor has also shown that two reporter genes can be used in live animals to image and measure two events. For example, both Flue and Rluc (or hRluc) can be inserted into a living animal, followed by administration of their respective substrates, to image two events, distinguishable by the fact that each has a specific substrate, and that the substrates do not cross-react with their non-partner enzyme. Similarly, the different kinetics of Rluc and hRluc relative to Flue permit the use of both enzymes in a living animal. In this case, two substrates are still used, but signal from Renilla luciferase decreases much more rapidly, so that imaging the animal at early time points and late time points allows one to distinguish the two signals. Signals are not distinguished due to wavelength differences, but due to requirement for two distinct substrates (D-luciferin and coelenterazine) and the kinetics of light production.

As the results herein show, the use of Renilla luciferase has several advantages over the use of FL when applied to living animals. One significant advantage of Renilla luciferase in living animals over firefly luciferase is that RL does not require ATP to produce light. All that RL requires is its substrate, coelenterazine, and oxygen. Firefly luciferase, on the other hand, requires not only its substrate, D-luciferin, and oxygen, but also magnesium and ATP. Accordingly, only intracellular FL function can be studied as ATP is not available extracelluarly.

As has now been shown, however, non ATP-dependent Renilla luciferase can be used and detected in living organisms, where it can be used to monitor extracellular events such as ligand-receptor binding, cell-cell interactions, and the like. This is particularly significant because it allows the use of a reporter gene with substrate in a living animal using non ATP dependent light production, facilitating multiplexed studies and opening the door to many different applications. Also, the use of two distinct signals (one from each reporter FL and RL/hRL) allows the following of two independent events.

Applications for the use of Rluc and/or hRluc in living animals include, but are not limited to, the following:

1. Tracking gene delivery. Using techniques well known in the art, one can construct gene therapy vectors, such as those based on adenoviruses, retroviruses, or the like, but, in addition, insert the Rluc or hRluc reporter gene into these vectors. When the virus infects, the reporter gene will be delivered along with the therapeutic gene, allowing tracking of gene delivery.

2. Gene expression. As discussed above, using techniques known in the art, the Rluc or hRluc gene can be fused to a promoter of interest and implanted into a living organism to investigate in vivo gene expression, as monitored by production of bioluminescent lights following exposure to coelenterazine.

3. Transgenic mice. Transgenic mice can be constructed, using techniques known in the art, with Rluc or hRluc as the transgene, operably linked to an endogenous promoter, and/or operably linked to a second transgene. Thus, for example, one could fuse the promoter of a known cancer related gene to the Rluc reporter, such that when the cancer gene is transcribed, Rluc is also transcribed. The resultant production of bioluminescence following exposure to coelenterazine serves as an indicator that the particular cancer-related gene has been activated. Other uses of Rluc or hRluc as a reporter gene for endogenous genes will be appreciated by one of skill in the art. using a reporter gene that is possible.

4. Cell trafficking. The RluclhRluc reporter genes can be used to determine how cells move from one place to another. For example, cells at a tumor site can be labeled with Rluc, and their subsequent metastasis to the liver and elsewhere tracked by periodic imaging of bioluminescence produced following the repeated injection of coelenterazine.

5. Drug studies. As will be appreciated by the skilled practitioner, one can build Rluc or hRluc reporter systems in which the bioluminescent signal is decreased or increased if a drug comes and does something inside the cell as, for example, by modulating a protein- protein interaction assay.

As noted above, cells containing Rluc or hRluc may be administered to living animals by injection into the blood stream and/or peritoneum, or by subcutaneous implantation. Other modes of administration are apparent to the skilled practitioner.

It should be noted that because the Renilla luciferase protein is not dependent on ATP, one can also inject the protein directly into an animal (instead of the gene encoding for the protein), and then the protein can produce signal when coelenterazine is administered. For example, one could fuse the RL protein to a antibody or antibody fragment, allowing one to image the location of the antibody as it binds to a cell surface receptor. This can not be done with Firefly luciferase because ATP is not generally available outside the cell. One can link Renilla luciferase protein to many other carriers (e.g., nanoparticles, peptides, etc.) and monitor their movement.

It should also be noted that although the optical imaging of Renilla luciferase reporter gene expression has been described herein with reference to its use in mice, the methods are applicable to other animals as well. In general, the present imaging technique can be used with any animal up to about the size of a large rat. In addition, the present technique may be used with animals of any size, including those larger than mice and rats, so long as the bioluminescence can be detected. Generally, in larger animals bioluminescence can be detected from a source located within about 2 centimeters of the outer surface of the animal. In human applications, one can use the present invention for skin imaging, imaging inside the body intraoperatively or using an endoscope, etc.

As discussed above, such studies are possible for RL, but not for FL, because RL is not ATP dependent. So if the RL protein enters the bloodstream and subsequently binds to a cell, light will be emitted if the substrate is provided to the animal even in the absence of ATP.

Modifications of the RluclhRluc gene to lead to proteins that encode for light of different wavelengths can also be performed and/or through the use of coelenterazine analogs that shift the wavelength of emitted light. These approaches may be useful as red/near infra red wavelengths better traverse tissue and may go significantly beyond the approximately 2 cm limit.

While this invention has been described in detail with reference to a certain preferred embodiments, it should be appreciated that the present invention is not limited to those precise embodiments. Rather, in view of the present disclosure which describes the current best mode for practicing the invention, many modifications and variations would present themselves to those of skill in the art without departing from the scope and spirit of this invention. The scope of the invention is, therefore, indicated by the following claims rather than by the foregoing description. All changes, modifications, and variations coming within the meaning and range of equivalency of the claims are to be considered within their scope.

Claims

WHAT IS CLAIMED IS:

1. A noninvasive method for detecting the localization of a cell within an animal, comprising administering to the animal a transformed cell expressing Renilla luciferase ("RL"), after a period of time in which the transformed cell can achieve localization in the animal, administering coelenterazine to the animal, immobilizing the animal within the detection field of a photodetection device, and detecting light emission from RL localized in the animal.

2. The method of claim 1, wherein the animal is a mammal.

3. The method of claim 2, wherein the mammal is a rodent.

4. The method of claim 1, wherein the transformed cell contains the gene Rluc.

5. The method of claim 1, wherein the transformed cell contains a synthetic gene hRluc.

6. A noninvasive method for detecting the localization of an entity within an animal, comprising administering to the animal an entity conjugated to RL protein, after a period of time in which the conjugated entity can achieve localization in the animal, administering coelenterazine to the animal, immobilizing the animal within the detection field of a photodetection device, and detecting light emission from RL localized in the animal.

7. The method of claim 6, wherein the entity is a cell.

8. The method of claim 6, wherein the entity is an antibody.

9. The method of claim 6, wherein the entity is a polypeptide.

10. The method of claim 6, wherein the entity is a nanoparticle.

11. The method of claim 6, wherein the animal is a mammal.

12. The method of claim 11, wherein the mammal is a rodent.

13. The method of claim 11, wherein the mammal is a human.

14. A noninvasive method for detecting the level of expression of a gene of interest in an animal over time, comprising operably fusing a nucleic acid encoding RL to the gene of interest, administering the fused gene to the animal, administering coelenterazine to the animal, immobilizing the animal within the detection field of a photodetection device, and measuring the level of light emission from RL in the animal with the photodetection device at selected time intervals sufficient to detect changes in the level of light emission in the animal over time.

15. The method of claim 14, wherein the nucleic acid is Rluc.

16. The method of claim 14, wherein the nucleic acid is hRluc.

17. A method for detecting a promoter-induction event in an animal, comprising administering coelenterazine to a transgenic animal having a promoter responsive to the event and a heterologous gene encoding RL under the control thereof, triggering the event in the transgenic animal, and measuring with a photodetector device light emission through opaque tissue from the RL in the transgenic animal.

18. The method of claim 17, wherein the heterologous gene is Rluc.

19. The method of claim 17, wherein the heterologous gene is hRluc.

20. A noninvasive method for detecting the localization of a gene therapy vector within an animal, comprising operably fusing a nucleic acid encoding RL into the gene therapy vector, administering the fused gene therapy vector to the animal, after a period of time in which gene therapy vector can achieve localization in the animal, administering coelenterazine to the animal, immobilizing the animal within the detection field of a photodetection device, and detecting light emission from RL localized in the animal.

21. The method of claim 20, wherein the nucleic acid is Rluc.

22. The method of claim 20, wherein the nucleic acid is hRluc.

23. A method for detecting two independent promoter-induction events in an animal, comprising providing a transgenic animal having a first promoter responsive to the first event and a heterologous gene encoding RL under the control thereof and a second promoter responsive to the second event and a heterologous gene encoding FL under the control thereof, administering coelenterazine to the animal, administering D-luciferin to the animal, triggering the first event in the transgenic animal, triggering the second event in the transgenic animal, and measuring with a photodetector device light emission through opaque tissue in the transgenic animal.

24. The method of claim 23, wherein the heterologous gene is Rluc. .

25. The method of claim 23, wherein the heterologous gene is hRluc.