JP2009011318A - Non-invasive localization of light-emitting complex in mammalian - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a non-invasive method for detecting, positioning and tracing a pathogen in living bodies such as mammalians, and other materials. <P>SOLUTION: The non-invasive method is to detect expression of a heterogous gene in a living non-human mammalian object. The method includes a process for providing the non-human mammalian object having cells containing an introduced gene, wherein (i) the introduced gene contains a heterogous gene encoding bioluminescent proteins or fluorescent proteins and (ii) the object contains opaque tissues, and a process for detecting the expression of the heterogous gene by measuring photon irradiation passing through the opaque tissues by using a photodetector, wherein the object is maintained under a condition permitting the expression of the heterogous gene by the cells. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention relates to non-invasive methods and compositions for detecting, positioning and tracking illuminants and luminescent biological events in mammalian subjects.

Current in vitro methods for detecting and quantifying infectious agents in tissues have limited ability to monitor the progression of infectious diseases. Replication of infectious agents in the host often involves primary, secondary and tertiary replication sites. Replication sites and the process by which infectious agents pass through those sites are observed based on the inoculation route, host-encoded factors, and determinants of the infectious factors.

Possible replication sites and infection progression may be estimated from experience. However, the site of infection and the rate of disease are often unknown or can only be roughly estimated. Also, the progression of infectious disease is often individual, even in inbred mouse strains, to determine what the disease will average on the experimentally infected host, There is a need for continuous in vitro analysis of a large number of infected animals.

It would be desirable to have a means of tracking the progress of infection in animal models. It would be ideal if the tracking could be done non-invasively and an animal could be evaluated as frequently as needed without adverse effects. The present invention seeks to provide a non-invasive method for detecting, positioning and tracking pathogens and other objects in vivo such as mammals.

In one aspect, the present invention provides a non-invasive method for detecting the localization of a biocompatible material in a mammalian subject comprising the steps of: (a) providing the subject with the biocompatible material and a luminescent component. Administering a complex; (b) after a period of time that the complex can achieve localization within the subject; and immobilizing the subject within the detection field of the photodetection device; (c) placing the subject in a stationary state And (d) during maintenance, photon emission from a luminescent component localized in the object is measured with the photodetection device until an image of the photon emission can be constructed, and (e) the image Build up.

In one embodiment, the above method may further comprise repeating steps (b) to (e) at selected intervals, the iteration tracking the localization of the biocompatible material in the subject over time. Can be effective to do.

In another embodiment, the above method can be performed using a multiplying charge coupled photodetection element.

In another embodiment, the above method can be a method for detecting the localization of tumor cells in a mammalian subject, wherein the administration can comprise administration of a complex containing a tumor cell targeting moiety.

In another embodiment, the above method can be a method for detecting the location of inflammation in a mammalian subject, wherein the administration can comprise administration of a complex containing an inflammatory targeting component.

In another embodiment, the method described above can be a method for detecting the localization of an infection by a pathogen in a mammalian subject, wherein the administration can comprise administration of a complex containing an infection targeting component.

In another embodiment, the method described above can be a method for detecting the localization of thrombotic plaques in a mammalian subject, the administration of which can include administration of a complex containing a thrombotic plaquer targeting component.

In another embodiment, the above method may comprise administration of a complex whose administration is a particle containing a luminescent component.

In another embodiment, the above method can be a method for detecting the localization of an infection by a pathogen in an animal model, and the biocompatible can be the pathogen. In a preferred embodiment, the pathogen can be Salmonella.

In another embodiment, the luminescent component can be a luminescent protein. In a preferred embodiment, the protein may be selected from the group consisting of luciferase, yellow fluorescent protein and ferredoxin IV. In another preferred embodiment, the administration may comprise administration of a complex that is a fusion protein of an antibody fragment and a luminescent protein. In another preferred embodiment, the biocompatible product can be a transformed cell and the luminescent component can be the product of a heterologous gene expressed from the cell. In a further preferred embodiment, the expression of the heterologous gene can be under the control of an activatable promoter.

In another aspect, the invention is a non-invasive method for detecting the level of biocompatible material in a mammalian subject over time, comprising: (a) providing the subject with a complex of the biocompatible material and a luminescent component. (B) place the subject in the detection field of the light detection device;
c) maintaining the object in the detection field of the device, and (d) while maintaining, photon emission from the luminescent component in the object is measured with the light detection device and from (e) (b) to ( It provides a method that consists of repeating the steps up to d) at selected intervals, which is effective for detecting changes over time in the level of biocompatible material in the subject.

In a further aspect, the present invention provides a non-invasive method for detecting transgene integration in a mammalian subject comprising the steps of: (a) effectively integrating and introducing into the subject a mammalian cell. A vector construct containing a gene, a gene encoding a luminescent protein and an activatable promoter (provided that
The gene encoding the luminescent protein is under the control of the promoter), (b) after a period of time during which the construct can achieve integration, activate the promoter, and (c) detect the subject with a light detection device Placed in the field, (d) maintaining the object in the detection field of the light detection device, and (e) during the maintenance, the level of photon emission from the expressed photoprotein in the object Measured with a detector,
(F) If the level of photon emission is significantly higher than background, confirm the incorporation of the transgene.

In yet a further aspect, the present invention provides a non-invasive method for detecting the localization of a promoter-inducing event in an animal comprising the following steps: (a) an inducible promoter responsive to that event and under its control In a transgenic animal with a heterologous gene encoding a photoprotein, the event is triggered, (b) the animal is placed in the detection field of a light detector, and (c) the animal is kept stationary And
(D) during maintenance, photon emission from the expressed photoprotein localized in the animal is measured with the photodetection device until an image of the photon emission can be constructed,
(E) Build the image.

In yet another aspect, the present invention provides a Salmonella transformed with a gene that expresses a luminescent protein.

In one aspect, the invention encompasses a non-invasive method for detecting the location of a biocompatible within a mammalian subject. The biocompatible material may be a molecule, macromolecule, cell,
Microorganisms (including pathogens) or particles may be used.

In this method, a complex of the object and a luminescent component is administered to a subject. The luminescent component is typically a molecule or macromolecule (macromolecule) that emits light. This component can emit light as a result of radiation absorption (eg, a fluorescent or phosphorescent molecule) or emit light as a result of a chemical reaction (eg, a bioluminescent protein). Typical examples of luminescent components are bioluminescent proteins such as luciferase and aequorin and colored or fluorescent proteins such as yellow fluorescent protein and ferredoxin IV.

This component can be incorporated during the synthesis of the object (eg, chemical or genetic incorporation, antibody fragments and photoprotein fusion proteins, etc.), post-synthesis chemical coupling, non-covalent association (eg, encapsulation by liposomes) ), I in the object
n situ synthesis (eg, expression of heterologous bioluminescent protein in transformed cells), or in situ controlled promoter-regulated bioluminescent protein in cells of transgenic animals stimulated with a promoter inducer
It can be complexed with the object by various techniques such as expression (eg, an interferon-activated promoter stimulated by viral infection).

After a period in which the complex can be localized in the object, the detection field of the photodetector for a period of time that is effective for measuring the object with a sufficient amount of photon radiation (with its photodetector) to construct an image Secure inside. A typical photodetection device is a multiplying charge coupled device (ICCD) camera connected to an image processing device. If an image can be constructed in a short time compared to the time scale in which the “unfixed” object moves, the object is essentially “fixed” during imaging and no special fixing measures are required. . Next, an image is constructed from the photon emission data.

By repeating the imaging process at selected intervals and constructing an image corresponding to each interval, the localization of the object in the object can be tracked over time by the method described above.
The methods described above can be used for a number of specific applications by combining, combining, or incorporating targeting components on an object. The targeting component may be an intrinsic property of the object (eg, antibody or antibody fragment), or the targeting component may be complexed, bound, or incorporated into the object (eg, a liposome containing an antibody). Examples of targeting components include antibodies, antibody fragments, enzyme inhibitors, receptor binding molecules, various toxins and the like. Targets for targeting components include inflammatory sites, infected sites, thrombotic plaques, tumor cells and the like. Markers suitable for recognition by targeting components that characterize these targets are well known.

Furthermore, if a pathogen (for example, Salmonella) combined with a luminescent component is used as an object, the site of infection by the pathogen in the animal model can be detected and localized (localized) by this method.

As a related aspect, the present invention encompasses a non-invasive method for detecting the level of biocompatible material in a mammalian subject over time. This method is similar to the method described above, but does not necessarily localize the object in the form of an image, and is designed to detect changes in the level of the object in the object over time. This method is particularly useful for monitoring the effect of therapeutic substances (eg antibiotics) on the level of objects (eg luminescent bacteria) over time.

In another aspect, the invention encompasses a non-invasive method for detecting transgene integration in a mammalian subject. In this method, a vector construct effective to integrate the transgene into mammalian cells is administered to the subject. Such constructs are well known in the art. This construct contains, in addition to the elements necessary for effective integration, a transgene (eg, a therapeutic gene) and a gene encoding a luminescent protein under the control of a selected activatable promoter. After a period of time that the construct can achieve integration, the promoter is activated. For example, when an interferon promoter is used, interferon production can be stimulated by local administration (eg, footpad injection) of polyinosin-polycytosine duplex (poly IC). Next, the target is a photodetection device (eg photomultiplier)
Place in the detection field of night vision “goggles” and measure or evaluate the level of photon emission, if that level exceeds the background (ie light is preferentially detected in the “activated” area) If possible, the subject is recorded as incorporating the transgene.

As a related aspect, the present invention includes a non-invasive method for detecting the localization of promoter-inducible events in animals transgenic or chimeric with a construct comprising a gene encoding a luminescent protein under the control of an inducible promoter. To do.
For the promoter induction event, administration of a substance that directly activates the promoter, administration of a substance that stimulates production of an endogenous promoter activator (for example, stimulation of interferon production due to RNA virus infection), endogenous promoter activator For example, heat shock or stress. This event is triggered and animals are imaged as described above.

In a further aspect, the present invention provides a photoluminescent protein (eg, luciferase)
Pathogens transformed with a gene that expresses (eg, Salmonella).

In another aspect, the invention also encompasses a method for identifying therapeutic compounds that are effective in suppressing the spread of infection by pathogens. In this method, a complex of a pathogen and a luminescent component is administered to a control animal and an experimental animal, the experimental animal is treated with a therapeutic compound candidate, and a luminescent pathogen in the control animal and the experimental animal is localized by the method described above. A compound is recognized as being therapeutically effective if it has become effective and has a potency that significantly suppresses the spread or replication of pathogens in laboratory animals compared to control animals. Complexes include fluorescently labeled antibodies, fluorescently labeled particles, fluorescently labeled small molecules, and the like.

As a further aspect, the present invention encompasses a method for localizing an object conjugated with a luminescent component through a medium with varying opacity. In this method, a photon transmitted through the medium is detected using a photodetection device, the photon is integrated over time, and an image is created based on the integrated signal.

In a further aspect, the present invention relates to a selected substance (at a specific site in an organism (
For example, a method for measuring the concentration of dissolved oxygen or calcium) is included. The method includes an object (eg, a cell) that contains a concentration sensor (ie, a luminescent molecule whose ability to emit light depends on the concentration of the selected substance). The object containing the luminescent molecule is administered in a substantially uniform distribution within the animal or a particular tissue or organ system (eg spleen). The creature is imaged. The intensity and localization of the light emission correlate with the concentration and position of the selected substance. Alternatively, the object contains a molecule that can emit light at a different wavelength than the second label, eg, a concentration sensor. If the non-uniformity of the distribution of objects in the host is normalized using this second label, the concentration of the selected substance can be determined more accurately.

In another aspect, the invention encompasses a method for identifying therapeutic compounds that are effective in inhibiting tumor growth and / or metastatic spread. In this method, (i
) Administering tumor cells labeled with a luminescent component or tumor cells containing a luminescent component to a group of experimental animals and a control animal; (ii) treating the experimental group with the selected compound;
i) Localize the tumor cells in the animals of both groups by imaging photon emission from the luminescent molecules associated with the tumor cells with a photodetector, and (iv) experiment with the compound compared to the control group A compound is recognized as therapeutically effective if it can significantly inhibit tumor growth and / or metastatic spread in the group.

These and other objects and features of the invention will be more fully understood when the following detailed description of the invention is read in conjunction with the accompanying drawings.

That is, the present invention provides a non-invasive method for detecting, positioning and tracking pathogens and other objects in a living body such as a mammal.

(I. Definition)
Unless otherwise indicated, all terms used in this specification have their ordinary meanings used in the technical field of the invention.

As used herein, an opaque medium refers to a "conventional" opaque medium and is not necessarily completely opaque. Thus, an opaque medium refers to a medium that is generally considered neither transparent nor translucent, including wood chips, mammalian meat and skin.

Unless otherwise specified, luciferases include prokaryotic luciferases and eukaryotic luciferases, and also include variants with altered or altered optical properties such as luciferases that emit light in the red region of the wavelength.

Biocompatible refers to an object that can be administered to a mammal. This includes pathogens that can be harmful to mammals. In the case of an animal having a cell containing a transgene that expresses a photoprotein, a biocompatible product refers to a transgene-containing cell that constitutes the mammal.

  Luminescence means that light can be generated by chemical reaction or absorption of radiation.

In this specification, light is about 300 nm and about 1100 n unless otherwise specified.
Electromagnetic radiation with a wavelength between m.

Infection prevalence usually refers to pathogen transmission and colonization of host sites other than the original site of infection. However, the term can also include an increase in the size and / or number of pathogens at the initial site of infection.

  Prokaryotic genes related to lux-luciferase and photon emission.

  Eukaryotic genes involved in luc-luciferase and photon emission.

A promoter induction event refers to an event that results in direct or indirect induction of a selected inducible promoter.

A heterologous gene refers to a gene that has been transfected into the host organism. Typically, a heterologous gene refers to a gene that is not originally derived from the genomic DNA of a transfected or transformed cell.

(II. General overview of the invention)
The present invention includes methods and compositions relating to non-invasive imaging and / or detection of luminescent complexes in mammalian subjects. This complex contains a biocompatible material and a luminescent component. Biocompatible materials include small molecules such as cyclic organic molecules; macromolecules such as proteins;
Microorganisms such as viruses, bacteria, yeasts, molds; all types of pathogens and pathogens; including but not limited to particles such as beads and liposomes. In addition, the biocompatible body may be all or part of the cells constituting the mammalian object to be imaged.

Luminous ability is imparted to the object by complexing the luminescent components. Such components include fluorescent molecules, fluorescent proteins, enzymatic reactions that emit photons, and luminescent materials such as bioluminescent proteins. Conjugation can include chemical coupling, genetic manipulation of the fusion protein, or transformation of cells, microorganisms or animals that express the bioluminescent protein. For example, if the object is a cell that constitutes a mammalian object to be imaged, the luminescent component may be a bioluminescent protein or a fluorescent protein “conjugated” to the cell, which is a transgenic or chimeric animal Due to the localization of promoter-regulated expression from the vector construct introduced into the cell.

Typically, the luminescent complex is administered to the subject in various ways, localized within the subject, and imaged. Imaging or measurement of photon emission from an object may last for tens of minutes and is not always so, but usually the object is fixed during the imaging process.

Imaging of the illuminator requires the use of a photodetector that can detect very low levels of light (usually a single photon event) and integrate the photon emission until an image can be constructed. An example of such a sensitive photodetector is a camera or camera that can amplify single photon events and then detect single photons (eg, cooled with liquid nitrogen) against background noise inherent in the detection system. In groups, there are devices that detect the event.

Once a photon emission image has been created, it is typically used to provide a compliant coordinate system with respect to the source of emitted photons (ie, to localize the luminescent complex with respect to the object) and to “normal” reflections of that object. Superimpose on the light image. Then, by analyzing such “synthesized” images, the location and / or amount of the target within the subject is determined.

  In the following, the above steps and embodiments are described in more detail.

(III. Luminescent body)
(A. Luminescent component)
Light-generating components useful in the practice of the present invention
ety; LGM), luminescent molecules or luminescent constructs may take any of a variety of forms depending on the application. A common feature of these is that they are luminescent, ie from atoms or molecules to ultraviolet (UV) as a result of the transition from an electronically excited state to a lower energy state (usually the ground state). Radiating electromagnetic radiation in the visible and / or infrared (IR) region.

Examples of the luminescent component include light-cooled (photoluminescence) molecules such as fluorescent molecules, chemiluminescent compounds, phosphorescent compounds, and bioluminescent compounds.

Two features of LGM that are strongly relevant to the present invention are their size and spectral characteristics. These will be discussed in connection with the specific types of luminescent components described below after a general discussion on spectral characteristics.

(1. Spectral characteristics)
An important aspect of the present invention is the selection of a luminescent component that produces light that can penetrate the animal tissue so that it can be detected non-invasively from the outside. The ability of light to pass through a medium such as animal tissue (mostly water) depends mainly on the intensity and wavelength of the light.

The stronger the emission intensity in a unit volume, the easier it is to detect the light. The intensity of light generated in a unit volume depends on the spectral characteristics of individual LGMs described later and the concentration of the component in the unit volume. Therefore, for example, LGM is typically applied to each object by a conjugation method in which a high concentration of LGM is placed in or on the object (for example, high-efficiency loading of liposomes or high-level expression of bioluminescent protein in cells). A brighter-emitting conjugate (LEC) is created that is easier to detect through deeper tissue layers than a single conjugation method.

The second factor that governs the detectability of LGM across the tissue layer is the wavelength of the emitted light.
Since most tissues consist primarily of water, water can be used to approximate the absorption characteristics of animal tissues. It is well known that water allows easier transmission of long wavelength light (red region light) than short wavelength light.

Therefore, as a rule, an LGM that emits light in the yellow to red (550-1100 nm) range is preferred over an LGM that emits at shorter wavelengths. LG described later
Some of M radiates in this range. However, according to the later experiments conducted in support of the present invention, when the present invention is implemented with an LGM emitting in the 486 nm region, good results can be obtained despite the fact that it is not the optimum emission wavelength. it can. These results were attributed to the presence of a relatively high concentration of LGM (luciferase molecule) in the LEC (transformed Salmonella cells) used in these experiments and the use of a highly sensitive detector. It is thought that. It will be appreciated that if LGM with a more optimal emission wavelength is used, similar detection results can be obtained with LGE having a lower concentration of LGM.

(2. Fluorescent component)
Fluorescence is the emission of a substance resulting from a single electronically excited state with a very short duration after removal of the radiation source. Since a part of the excitation light is converted into heat by the fluorescent molecule, the wavelength of the emitted fluorescence is longer than the wavelength of the excitation light (Stokes' law).

The use of fluorescent molecules in the present invention will be more complicated than the use of bioluminescent molecules, since it requires light input for the fluorescent molecules to exhibit luminescence. Typically, precautions are taken to shield the excitation light so as not to contaminate the fluorescent photon signal detected from the subject. A self-evident precaution is to install an excitation filter, such as that used in a fluorescence microscope, in the radiation source. A properly selected excitation filter blocks most of the photons having a wavelength similar to that of the photons emitted by the fluorescent component. Also, a barrier filter is used in the detector to screen out most of the photons having wavelengths other than those of the fluorescent photons. The filter as described above is
Available from a variety of commercial sources including mega Optical (Brattleboro, VT).

It is also possible to excite the fluorescent component using a laser that generates high intensity light that is close to the appropriate excitation wavelength and not close to the fluorescence emission wavelength. For example, an xy conversion mechanism may be used so that the laser can scan the object as in a confocal microscope.

As another precaution, a radiation source can be placed behind the object and shielded so that only those photons that have reached the detector site have completely passed through the object. In addition, a detector with low sensitivity to the wavelength of light used to excite the fluorescent component may be selected.

If the above measures are applied wisely, it is possible to detect fluorescent LGM by the method of the present invention.

Fluorescent components include small fluorescent molecules such as fluorescein and green fluorescent protein (Chalfi et al., 1994, Science 263: 802-80.
5; Morin and Hastings, 1971, J. MoI. Cell. Physi
ol. 77: 313), lumazine and yellow fluorescent protein (O'Kane et al.,
1991, PNAS 88: 1100-1104; Daubner et al., 1987.
, PNAS 84: 8912-8916). In addition, certain colored proteins such as ferredoxin IV (Grabau et al., 1991, J. Biol. Chem. 266: 3294-3299; their fluorescence has not yet been evaluated) are also fluorescent, and the present invention. May be available at. Ferredoxin IV is a particularly promising candidate because it has a reddish color, which can fluoresce or reflect at relatively long wavelengths, producing light that is effective to penetrate tissue. Furthermore, since this molecule is small as a protein (95
Amino acids), when conjugated to an object, the effect on their function can be minimized.

The advantage of a small fluorescent molecule is that it is less likely to interfere with the biological activity of the object to which it is attached than a larger luminescent component. Commercially available fluorescent molecules with various excitation and emission spectra suitable for use in the present invention can also be obtained. For example, Molecular Probes (Eugene, OR) sells a number of fluorophores including Lucifer Yellow (absorbing at 428 nm and emitting at 535 nm) and Nile Red (absorbing at 551 nm and emitting at 636 nm). In addition, molecules derivatized with various groups that can be used in various conjugation methods are also available (eg, from Molecular Probes).

(3. Bioluminescent components)
Chemiluminescence (luminescence as a result of chemical reaction) and bioluminescence (visible luminescence from the living body)
Have been studied in detail from many aspects (eg Campbell, 1988).
, Chemiluminescence. Principles and Ap
applications in Biology and Medicine (Chichester, UK; Elis Horwood and VCH publishers)). The salient features are briefly summarized below.

Bioluminescent molecules differ from fluorescent molecules in that they do not require input of radiant energy to emit light. Rather, bioluminescent molecules generate light using chemical energy such as ATP. The advantage of a bioluminescent component over a fluorescent component is that there is virtually no background in the signal. The only light detected is light generated by an exogenous bioluminescent component. In contrast, light used to excite fluorescent molecules often results in fluorescence of substances other than the intended target. This is especially true when the "background" is as complex as the living animal's internal environment.

Several types of bioluminescent molecules are known. They include the luciferase family (eg Wood et al., 1989, Science 244: 700
-702) and the aequorin family (eg, Prasher et al., Bioche)
m. 26: 1326-1332). The elements of the luciferase family are
It has been identified in various prokaryotes and eukaryotes. Luciferase and other enzymes involved in the prokaryotic luminescence (lux) system, and the corresponding lux gene, are vibrio (V
It has been isolated from marine bacteria of the genus ibrio and Photobacterium and terrestrial bacteria of the genus Xenorhabdus.

A representative eukaryote containing the luciferase system (luc) is the North American firefly Photinus pyralis. Firefly luciferase has been studied in detail and is widely used in ATP assays. A kind of click beetle Py
A cDNA encoding luciferase from Rophorus plagiophthalamus has been cloned and expressed (Wood et al., 198).
9, Science 244: 700-702). This beetle is unusual in that different species of species emit different colors of bioluminescence. Four clones with 95-99% homology to each other were isolated. They are 546nm (green), 560nm (
Yellow-green), 578 nm (yellow), and 593 nm (orange) light. The last type (593 nm) is particularly advantageous for use as a luminescent component in the present invention because its emitted light has a wavelength that penetrates tissue more easily than shorter wavelength light.

Luciferases and aequorin-like molecules are derived from energy sources such as ATP and NAD (P) H and luciferin or coelentridine.
e) and a substrate such as oxygen is required.

In order for the luciferase enzyme to emit light, the substrate luciferin must be supplied to the luciferase enzyme. When the luciferase enzyme is introduced as an expression product of a vector containing a cDNA encoding lux luciferase, a convenient way to supply luciferin is not only to express luciferase, but also to include a biosynthetic enzyme for synthesizing luciferin. It is to express. In cells transformed with such a construct, oxygen is the only exogenous element required for bioluminescence. Using such a method (detailed in Example 1), a lux transformed Salmonella is generated and used to perform the experiments described below to support the present invention.

Plasmid constructs encoding the lux operon from the soil fungus Xenorhabdus luminescens (Fraccman et al., 1990, J. Ba
ct. 172: 5767-5773) confers photon radioactivity on transformed E. coli by expressing two subunits of heterodimeric luciferase and three auxiliary proteins (Fraccman et al., 1990). X. lumine
Optimal bioluminescence of E. coli expressing the lux gene of scens was observed at 37 ° C. (Szittner and Meichen, 1990, J. Biol. Ch.
em. 265: 16581-16687; Xi et al., 1991, J. MoI. Bact. 1
73: 1399-1405), in contrast to the low optimum temperature of luciferases from eukaryotic and other prokaryotic organisms (Campbell, 1988, C).
hemilescence. Principles and Appl
ics in Biology and Medicine (Chichester, UK: Ellis Horwood and VCH Publisher)). Therefore, X. The luciferase from luminescens is suitable for use as a marker for studies in animals.

Luciferase vector constructs as described above or as described in Example 1 can be used to transform a variety of host cells including most bacteria and many eukaryotic cells (luc constructs). Also, certain viruses such as herpes virus and vaccinia virus can be genetically engineered to express luciferase. For example, Kovacs Sz. And Mettenlieter, 1991, J. et al. G
en. Virol. 72: 2999-3008 describe the stable expression of the gene encoding firefly luciferase in herpesviruses. Bra
sier and Ron, 1992, Meth. in Enzymol. 216:
386-396 describes the use of luciferase gene constructs in mammalian cells. Luciferase expression from cultured mammalian cells is macroscopically determined by CCD imaging (Israel and Honigman, 1991, Gene 104: 1
39-145) also microscopically (Hooper et al., 1990, J. Biolum.a).
nd Chemilum. 5: 123-130).

(B. Object)
The invention encompasses objects modified or conjugated to include luminescent components, luminescent constructs or luminescent molecules as described above. Such conjugates or modifications,
It is called a luminescent material, a luminescent complex (LEC) or simply a complex. These objects themselves can take the form of molecules, macromolecules, particles, microorganisms or cells, for example. The method of conjugating a luminescent component to an object depends on the component and the nature of the object. A typical conjugation method will be discussed in connection with the objects described below.

(1. Small molecule)
Small molecule objects that may be useful in the practice of the present invention include compounds that interact specifically with pathogens or endogenous ligands or receptors. Examples of such molecules include drugs and therapeutic compounds; toxins such as those present in the venom of toxic organisms including certain spiders, snakes, scorpions, dinoflagellates, marine snails, bacteria; NGF, PD
Examples include, but are not limited to, growth factors such as GF, TGF, and TNF; cytokines; bioactive peptides.

These small molecules are preferably conjugated (coupled) with a luminescent component (eg, a small fluorescent molecule (described above)) that, if any, interferes with the biological activity of the small molecule. The nature of the bond is usually chemical and may be done in any of a variety of ways known to those skilled in the art.

Small molecule objects can be synthesized to contain a luminescent component so that a formal binding operation is not required. It is also possible to synthesize a small molecule object having a reactive group capable of reacting with a luminescent component and vice versa.

Small molecules conjugated to luminescent components of the present invention may be used in animal models of human diseases or conditions or directly in the human subject to be treated. For example, a small molecule that binds with high affinity to a receptor expressed on tumor cells can be used in animal models to localize the tumor and obtain an estimate of its size, or a tumor after treatment with a therapeutic drug candidate Changes in growth or metastasis of can be monitored. Such molecules can also be used to monitor tumor characteristics as described above in cancer patients.

(2. Polymer)
Macromolecules such as polymers and biopolymers are another example of objects useful in the practice of the present invention. Exemplary macromolecules include antibodies, antibody fragments, fusion proteins, and certain vector constructs.

Purchased from commercial sources or known in the art (Harlow et al.,
1988, Antibodies: A Laboratory Manual,
Using antibodies or antibody fragments created by Chapter 10, page 402, Cold Spring Harbor Press), these antibodies are combined with a luminescent component,
By administering the complex to the subject, for example, by injection, localizing the complex at the site of the antigen, and imaging the complex, the antigen in the mammalian subject can be localized.

Antibodies and antibody fragments have several advantages for use as objects in the present invention. These, by their nature, themselves constitute a targeting component. Furthermore, because of its size, it is easily adapted to binding with several types of luminescent components including small fluorescent molecules, fluorescent proteins and bioluminescent proteins, and can diffuse more rapidly than, for example, cells and liposomes.

The luminescent component can be directly bound to the antibody or fragment, or can be indirectly bound using, for example, a fluorescent secondary antibody. Direct conjugation can be performed by general chemical conjugation of, for example, a fluorophore to the antibody or antibody fragment, or by genetic engineering. Chimeric or fusion proteins can be constructed that contain antibodies or antibody fragments conjugated to fluorescent or bioluminescent proteins. For example, Casadei et al., 1990, PANS 87: 2.
No. 047-2051 describes a method for preparing a vector construct capable of expressing an aequorin-antibody fusion protein gene in mammalian cells.

Complexes containing antibodies can be used in many applications of the present invention. For example, using a labeled antibody against E-selectin expressed at the site of inflammation, localizing the inflammation,
The effects of anti-inflammatory drug candidates can be monitored.

The vector construct itself can also be a polymer that can be applied to the present invention. For example, a gene encoding a luminescent molecule and a therapeutic gene are selected from a selected promoter (
That is, it is possible to construct a eukaryotic expression vector contained under the control of a promoter expressed in the target cell of the therapeutic gene. The expression of the luminescent molecule assayed using the method of the invention can be used to determine the expression location and expression level of the therapeutic gene. This method may be particularly useful when the expression of the therapeutic gene does not have a direct phenotype within the person or animal model being treated.

(3. Virus)
Another object useful in certain aspects of the present invention is a virus. Since many viruses are pathogens that infect mammalian hosts, they can be conjugated to luminescent components and used to study the site of initial infection and the spread of infection.
A virus labeled with a luminescent component can also be used for screening for drugs that suppress infection or the spread of infection.

Viruses can be labeled indirectly using antibodies conjugated to luminescent components or, for example, viral particles (Dhawan et al., 1991, J. Immunol. 14).
7 (1): 102) and the like, and after biotinylation, these are detectable components (
For example, it can be indirectly labeled by exposure to streptavidin conjugated to a fluorescent molecule.

Alternatively, the viral particles are made with a fluorophore such as rhodamine, eg, Fan et al., 1992, J. MoI. Clin. Micro. 30 (4): 905 may be used for direct labeling. Viruses can also be genetically engineered to express luminescent proteins. The genomes of certain viruses, such as herpes and vaccinia, are large enough to accommodate genes as large as the lux and luc genes used in experiments conducted in support of the present invention.

By using the labeled virus in an animal model, infection can be localized, the progress of the infection can be monitored, and drugs effective to suppress the spread of the infection can also be screened. For example, herpes virus infections appear as skin lesions, but the virus can also cause herpes encephalitis. By using viruses labeled with any of the methods described above, such infectious diseases can be localized and monitored, and various antiviral agents can be found in the central nervous system (C
NS) can be examined for its efficacy in infectious diseases.

(4. Particles)
Particles including beads, liposomes and the like constitute another object useful in the practice of the present invention. Since these are large in size, a larger number of light-emitting components can be conjugated to the particles than in the case of small molecules. This results in a higher concentration of light emission that can be detected with shorter exposure times or through a thicker tissue layer. Liposomes can also be constructed to include essentially pure targeting components or ligands (eg, antigens and antibodies) on their surface. Furthermore, for example, bioluminescent protein molecules can be loaded on liposomes at relatively high concentrations (Campbell, 1988, Chemiluminesc).
ence. Principles and Applications in
Biology and Medicine (Chichester, UK: Ellis)
Horwood and VCH publishers)).

In addition, the two liposomes can be directed to the same cell type so that light is produced only when both are present. For example, one liposome can carry luciferase and the other can carry luciferin. These liposomes may retain a targeting component, and the targeting components on the two liposomes may be the same or different. Viral proteins on infected cells can be used to identify infected tissues or organs. Cells of the immune system can be localized using single or multiple cell surface markers.

In order to prolong blood circulation time and enhance targeting by blood flow, it is preferable to coat the surface of the liposome, for example, by incorporating a phospholipid-polyethylene glycol complex. This type of liposome is well known.

(5. Cells)
Cells, both prokaryotic and eukaryotic, constitute another object useful in the practice of the present invention.
Cells can be loaded with a relatively high concentration of luminescent components as well as particles, but cells have the advantage that they can be supplied with luminescent components, for example, by transfecting cells with a heterologous gene construct. It is also possible to select cells that express a “targeting component” or a molecule that is effective to direct the cells to a desired location within the subject. Alternatively, cells can be transfected with a vector construct that expresses the appropriate targeting moiety.

The cell type used depends on the application. For example, as described in detail later, by using bacterial cells such as Salmonella, it is possible to study the infection process at a high level of temporal and spatial resolution and evaluate the effect of drugs or therapeutic agents on the infection process. it can.

Bacterial cells constitute an effective object. For example, by transfecting them, a high level of luminescent component or a high level of targeting protein can be easily expressed. It is also possible to identify colonies that express an antibody against a selected antigen by obtaining an E. coli library containing bacteria that express a surface-bound antibody and screening it (Strat).
agene, La Jolla, California). Next, the bacterium obtained from this colony is transformed with a second plasmid containing the gene for the luminescent protein, and the transformant is used in the method of the present invention as described above, so that The antigen can be localized.

By linking pathogenic bacteria to luminescent components and using them in animal models, the infection process can be tracked in vivo, or potential anti-infective drugs such as new antibiotics can be used to control the infection. Can be evaluated for efficacy. An example of this application will be described by an experiment described later as support for the present invention.

Eukaryotic cells are also useful as objects in certain aspects of the present invention. Appropriate expression vectors containing the desired regulatory elements are commercially available. Using these vectors, a construct capable of expressing a desired luminescent protein in various eukaryotic cells including primary cultured cells, somatic cells, lymphocytes and the like can be prepared. These cells can be used for transient expression tests, or in the case of cell lines, can be selected as stable transformants.

The expression of the luminescent protein in the transformed cells can be regulated using any of a variety of selected promoters. For example, if it is desired to use the cell as a illuminant that directs the cell to a certain site in the subject by the expressed ligand or receptor, a constitutively active promoter such as a CMV promoter or SV40 promoter may be used. Cells transformed with such a construct are
It can also be used to assay compounds that inhibit light generation, for example, by killing the cells.

In addition, the transformed cells should be administered so that the luminescent protein is expressed only under certain conditions, such as when the transformed cells are evenly distributed within the subject and subjected to viral infection or cytokines. Also good. Promoters that respond to these and other stimuli-related factors are known in the art. Related aspects include the Tet system (Gossen and Bujard, 1992, PANS
89: 5547-5551) can also be used to temporarily activate the expression of luminescent proteins.

For example, CD4 + lymphocytes can be transformed with a construct containing a tat-reactive HIV LTR element and used as an assay for HIV infection (I
srael and Honigman, 1991, Gene 104: 139-1.
45). Cells transformed with such constructs were transformed into SCID-hu mice (McC
une et al., 1988, Science 241: 1632-1639) and can be used as a model for human HIV infection and AIDS.

For example, tumor cell lines transformed as described above with a constitutively active promoter can be used to monitor tumor growth and metastasis. Tumor size and metastasis can be monitored while the transformed tumor cells are injected into an animal model to form a tumor mass and treated with a growth inhibitor candidate or a metastasis inhibitor candidate.

Tumor cells can also be generated from cells transformed with a construct containing a regulatable promoter with activity sensitive to various infectious agents or therapeutic compounds.

(6. Cell transformation)
Both prokaryotic and eukaryotic transformation methods are well known in the art (Sa
mbrook et al., 1989, Molecular Cloning: A Lab.
organization Manual, Cold Spring Harbor La
(Boratory Press, Volume 2). Vectors with appropriate regulatory elements and multiple cloning sites are widely available commercially (eg, Stratagene, La Jolla, California, Clontech, Palo Alto, Calif.).
(IV. Transgenic animal containing gene encoding photoprotein)
In another aspect, the present invention includes a transgenic animal containing a heterologous gene construct encoding a luminescent protein or luminescent protein complex. This construct is driven by the selected promoter and may contain, for example, various auxiliary proteins, selectable markers, enhancer elements, etc. necessary for the functional expression of the luminescent protein.

Activation of the promoter increases the expression level of the gene encoding the luminescent molecule and the auxiliary protein. Activation of the promoter is accomplished by interaction of the promoter element with the selected biocompatible or part thereof. If activation occurs only in a part of the animal, only that part of the cell will express the photoprotein.

For example, by using an interferon-inducible promoter (such as a 3′-5 ′ polyA synthase promoter), infection of transgenic cells with a number of different RNA viruses can be detected.

As a related aspect, a promoter that is expressed in a certain disease state can be used to label the affected area in the transgenic animal and the expression of the luminescent component can be used to monitor the effect of the treatment on that disease state. For example, E-selectin is expressed at sites of inflammation in vivo (Pober and Cotran, 1991, La
b. Invest. 64: 301-305). Thus, the E-selectin promoter can be isolated and used to drive the expression of the luciferase gene.

It is also possible to use the method of the invention with tissue specific promoters. This allows the screening of compounds that are effective in inhibiting pathogenic processes that cause, for example, degeneration of specific organs or tissues in the body, and also allows for the tracking of cells (eg neurons) in developing animals and the like .

Many promoters that can be used in the present invention are known in the art. In addition, in order to isolate a promoter-containing genomic DNA, a method for isolating a promoter of a cloned gene using information obtained from the cDNA of the gene is also known.

(V. Imaging of luminescent complex)
The luminescent complex localized at the intended site in the subject can be imaged in several ways. Such imaging guidelines and specific examples are described below.

(A. Localization of luminescent complex)
For a “targeted” object, ie, an object that contains a targeting component (a molecule or feature designed to localize an object to a specific site or sites within an object or animal), localization and Refers to a state in which an equilibrium is essentially established between a “localized” object bound in the object and an “free” object unbound. The rate at which such equilibrium is reached depends on the route of administration. For example, a complex administered by intravenous injection to localize a thrombus will achieve localization or accumulation in that thrombus within minutes after injection. On the other hand, complexes administered orally to localize intestinal infections will require several hours to achieve localization.

The term localization may also simply refer to the location of the object in the subject or animal at a selected time after administration of the object. For example, in the experiments detailed herein, Salmonella is administered (eg orally) and its spread is tracked as a function of time. In this case, the object can be “localized” immediately after oral administration, as long as it marks the initial location of the bacteria it has administered and subsequent spread or regression can be followed by imaging.

As a related aspect, the localization of injected tumor cells, e.g. expressing a luminescent component, will consist of the colonization of these cells at a site within the animal to form a tumor mass.

As another example, localization is achieved when the object becomes distributed after administration. For example, in the case of a complex administered to measure oxygen levels in various organs throughout the subject or animal, the complex will be released when it reaches an essentially steady state of distribution within the subject or animal. , "Localized" or beneficial.

In any of the above cases, one of ordinary skill in the art can appropriately estimate the time to achieve localization. The state of localization as a function of time can also be tracked by imaging the luminescent complex according to the method of the present invention.

(B. Photodetector)
An important aspect of the present invention is that a faint light from within a mammal can be imaged in a reasonable amount of time (preferably within about 30 minutes) and is sufficient to construct an image using the signal from the device. This is the selection of a highly sensitive photodetection device.

"Night vision" if a very bright luminescent component can be used and / or if it is possible to detect a luminescent complex localized near the surface of the subject or animal being imaged
Common high-sensitivity video cameras such as goggles and silicon multiplier (SIT) cameras (for example, Hamamatsu Photonic Systems, Bridgewater, NJ) can be used.
However, more generally, a more sensitive light detection method is required.

At very low light levels as encountered in practicing the present invention, the photon flux per unit area is so low that the object to be photographed seems no longer continuous. Instead, it is represented by individual photons that are independent of each other in time and space. When viewed on a monitor, such an image looks like a spot of blinking light, each representing a single photon detected.

By accumulating these detected photons over time in a digital image processing device, an image can be acquired and constructed. In contrast to conventional cameras that assign intensity values to the signal at each image point, the intensity of the signal has no meaning in the photon counting imaging method. Its purpose is simply to detect the presence of a signal (photon) and to count the number of occurrences of the signal over time with respect to its position.

Individual photons can be detected by at least two types of photodetectors described later, and a signal that can be analyzed by the image processing apparatus can be created.

(1. Noise reduction type photo detector)
The first type is a device that obtains sensitivity by reducing background noise in the photon detector as opposed to amplifying the photon signal. Noise is reduced primarily by cooling the detector array. This device has a “thin back”
A charge coupled device (CCD) camera called a cooled CCD camera is included. In a more sensitive apparatus, cooling is performed using, for example, liquid nitrogen that lowers the temperature of the CCD array to about −120 ° C. “Back thin” refers to an ultra-thin back plate that increases quantum efficiency by shortening the optical path length that is taken to detect photons. A particularly sensitive back-thin low-temperature CCD camera is a Photometr.
TE series 200 camera "TE" available from ics (Tucson, Arizona)
CH 512 ".

(2. Photon amplification device)
The second type of high sensitivity photodetector includes a device that amplifies photons before they hit the detection screen. This type includes CCD cameras with a multiplier such as a microchannel multiplier. Microchannel multipliers typically contain a metal array of channels that are perpendicular to and coextensive with the detection screen of the camera. The microchannel array is placed between the sample, object or animal to be imaged and the camera. Most of the photons that enter the channels of this array come into contact with the sides of the channels before exiting the channels. The voltage applied across the array is
Many electrons are emitted from each photon collision. Electrons resulting from such collisions escape from the channel in which they are generated in a “shotgun” fashion and are detected by the camera.

Higher sensitivity can be obtained if the multiplying microchannel array is placed in series so that the electrons generated in the first stage will then give the signal of the electrons amplified in the second stage. However, increasing sensitivity sacrifices spatial resolution, which decreases with each additional amplification step.

A typical example of a single photon detector of the microchannel multiplier type is Hamamat.
C2400 series available from su.

(3. Image processing device)
The signal generated by the light detection device that counts photons needs to be processed by an image processing device, for example, to construct an image that can be displayed on a monitor or printed by a video printer. Such image processing devices are typically sold as part of a system that includes the high-sensitivity photon counting camera described above, and thus the same supplier (
For example, it can be obtained from Photometrics and Hamamatsu).
Image processing devices obtained from other vendors can also be used, but generally more effort is required to build a functional system.

The image processing apparatus is usually an IBM compatible PC or Apple Macintosh (Appl).
e Connect to a personal computer such as Computer, Cue Party, CA. This personal computer may or may not be included as part of the purchased imaging system. Once the image is in the form of a digital file, it can be converted into various image processing programs (
For example, it is possible to operate and print with Adobe Systems, Inc. (Mountain View, Calif. “ADOBE PHOTOSHOP”).

(C. Fixed object in the detection field of the device)
(1. Device detection field)
The detection field of the device refers to the area where a consistent measurement of photon emission is obtained. For a camera that uses an optical lens, the detection field is simply the field of view that the lens gives to the camera. Also, if the light detection device is “night vision” goggles, the detection field is the field of view of the goggles.

The detection field may also be a surface defined by the ends of optical fiber cables arranged in a closely packed array. The array is constructed to maximize the area covered by the end of the cable (as opposed to the gap between the cables)
Installed very close to the subject. For example, a transparent material such as plexiglass can be installed adjacent to the object, and the array can be fixed from the opposite side of the object adjacent to the transparent material.

The end of the fiber optic cable on the opposite side of the array can be directly connected to a detector or multiplier, such as the input end of a microchannel multiplier, thus eliminating the need for a lens.

The advantage of this method is that photon scattering and / or loss is reduced by eliminating most of the air gap between the control and detector and / or by eliminating the lens. 60 mm A used in the experiments conducted as the backing of the present invention
Even a highly transmissive lens, such as an F Nikkor macro lens, transmits only a portion of the light that reaches the front lens element.

For stronger LGMs, a photodiode array can be used to measure photon emission. By incorporating the photodiode array into a relatively flexible sheet, the practitioner can partially “wrap” the array around the object. This method also minimizes photon loss and provides a means for obtaining bioluminescent three-dimensional images.

A three-dimensional image may be created using a method in which a plurality of detectors are installed around the object, or other methods including a scanning type detector or a group of detectors.

It will be appreciated that the entire animal or subject need not be within the detection field of the light detection device. For example, when measuring a luminescent complex that is known to be localized to a specific region of interest, measure only light from that region and its surrounding "dark" bands to obtain the desired information. All you need is enough.

(2. Fixing the target)
When it is desired to create a two-dimensional or three-dimensional image of an object, the object may be fixed in the detection field of the light detection device while measuring photon emission. If the signal is so bright that an image can be constructed from photon emissions measured in less than about 20 milliseconds and the object is not particularly upset, it ensures that the object is in the field of the detector at the start of the measurement period In addition, no special fixing measures will be necessary.

On the other hand, when the photon emission measurement requires about 20 msec or more and the target is shaken, the photon is adjusted to the degree of the shake of the target so that the spatial information is stored in the constructed image. During radiation measurements, precautions must be taken to ensure that the subject is not stationary. For example, if the subject is a person and the photon emission measurement time is on the order of a few seconds, it may be necessary to ask the subject to remain as still as possible during the photon emission measurement (imaging). On the other hand, when the object is an animal such as a mouse, the object can be fixed using, for example, anesthesia or a mechanical restraint tool.

Various restraints can be created. For example, a plexiglass sheet can be secured on a foam cushion to make a restraint effective for securing a mouse for tens of seconds to minutes. The cushion has an indentation for the animal's head at one end. The animal is placed under the plexiglass so that its head is over the indentation and breathing is free, but its body movement is constrained by a foam cushion.

If it is desired to measure only the total amount of light emitted from a subject or animal, it is not necessary to fix the subject even if the photon emission measurement period is long. All that is necessary is to keep the object confined to the detection field of the photodetector during imaging. However, if the subject is fixed during such a measurement, the thickness of the tissue through which the detected photons pass will be more uniform between animals, thus improving the consistency of the results obtained. It will be understood.

(D. Other problems during imaging)
(1. Fluorescence generating component)
In order to visualize the fluorescence generation component, an excitation light source is required together with a photodetector. It will also be appreciated that the excitation light source is lit while measuring photon emission from the luminescent component.

Appropriate selection of fluorophores, installation of light sources, and selection and installation of filters all facilitate the construction of information-rich images, as described above in the section on fluorescence generating components.

(2. High resolution image)
Light scattering by the organization limits the resolution that can be obtained with LGM imaging by measuring total photon emission. The present invention can focus on selected points in the subject,
It will be understood that it also encompasses aspects in which higher resolution images can be constructed by synchronizing the LGM emission to an external source that is not significantly scattered within the tissue. For example, an object to be imaged can be scanned three-dimensionally using a focused ultrasonic signal. Light emission from a region within the focal point of the ultrasound can be separated from other photon radiation by characteristic vibration imparted to the light by the ultrasound (eg, Houston and Moerner, US Pat. No. 4,614). , 11
No. 6, issued September 30, 1986)
(E. Construction of photon emission image)
If the image was obtained using “night vision” goggles or a high-sensitivity video camera because the luminescent component is exceptionally bright and / or because the luminescent complex is localized near the surface of the object, simply Simply view the image or display it on a video monitor. If desired, an image processing device that can store individual video frames in memory for analysis or printing and / or digitize the images for analysis and printing on a computer can bypass the signal from the video camera. Can do.

Also, when using the photon counting method, an array of numbers representing the number of photons detected at each pixel location is generated in the image processing device by measuring photon emission. These numbers are usually used to normalize the number of photons (with respect to a certain pre-selected value or the maximum number detected at any pixel), and then the normalized number to brightness (
Create an image by converting it to grayscale) or color (pseudocolor) and displaying it on the monitor. In the case of pseudo color display, typical color assignments are as follows. A pixel with zero photon count is assigned black, a lower count is assigned blue, a longer wavelength is assigned as the count increases, and a highest photon count is assigned red. The position of the color on the monitor represents the distribution of photon radiation and thus the position of the luminescent complex.

In order to provide a compliant coordinate system for the complex, one typically constructs a grayscale image of the object whose photon emission has been measured (still fixed). Such an image
For example, open the door toward the imaging room or imaging box in a faint room light,
It can be constructed by measuring the reflected photons (typically a time corresponding to a fraction of the time taken to measure the photon emission). This grayscale image may be constructed before measuring photon emission, or after measuring photon emission.

Typically, a composite image of the photon emission for the object is created by superimposing the photon emission image on the grayscale image.

If you want to track the localization of a luminescent complex and / or the signal from the luminescent complex over time, for example to record the effect of a treatment on the distribution and / or localization of a selected biocompatible component, By repeating photon emission measurement or imaging at selected time intervals, a series of images can be constructed. The interval may be as short as a few minutes or as long as a few days or weeks.

(VI. Analysis of photon emission images)
Images created using the methods and / or compositions of the present invention can be analyzed in various ways. It varies from simple visual inspection, brain assessment and / or hard copy printing to complex digital image analysis. The interpretation of the information obtained by analysis depends on the phenomenon to be observed and the object used.

The following experiment is one of the applications of the present invention (tracking Salmonella infection in living rats) and illustrates how the image obtained by the method of the present invention can be analyzed.

(VII. Imaging of Luminescent Salmonella in Living Mice)
In experiments conducted in support of the present invention, S. typhimurium (S
Characterize the distribution of Almonella typhimurium infection (animal model of human typhoid). Mouse pathogenic S. typhimurium SL1344 (Hoise
th and Stocker, 1981, Nature 291: 238-239
), SL1344 non-invasive mutant BJ66, and Salmonella low pathogenic LT-2 strain LB5000, each labeled with a plasmid containing the lux operon and used in experiments to localize Salmonella infection in mice.

(A. Construction of luminous Salmonella)
(1. Salmonella strain)
Three strains of Salmonella typhimurium with different pathogenic phenotypes revealed by oral and intraperitoneal inoculation of mice were selected for transformation.

The most pathogenic phenotype used here is SL1344, which is a mouse strain originally obtained from a calf's lethal infection (Hoiseth and St
ocker, 1981, Nature 291: 238-239). When mice are orally inoculated with this strain, the bacteria metastasize throughout the lymph system, resulting in liver, spleen and bone marrow colonization (Carter and Collins, 197).
4, J. Exper. Med. 139: 1189-1203; Finlay and Falkow, 1989, Mol. Microbiol. 3: 1833-18
41 and Hsu, 1989, Microbiol. Rev. 53: 390-409
See also the review).

A non-invasive mutant BJ66 of SL1344 is also evaluated. Oral inoculation of BJ66 usually does not cause systemic infection in mice, but systemic infection occurs when this strain is inoculated intraperitoneally.

The Salmonella low pathogenic LT-2 strain LB5000 is also examined. The LT-2 strain is a laboratory strain that is known to have reduced or variable pathogenicity to mice. LB5000 contains multiple auxotrophic mutations, is streptomycin resistant and is cleared from mice after oral or intraperitoneal inoculation.

(2. Transformation of Salmonella strain by lux operon)
Each of the above three strains is a plasmid encoding the lux operon in Example 1.
Transform as detailed below. Soil fungus Xenorhabdus lumine
This plasmid, obtained from scens (Fracman et al., 1990)
Two subunits of heterodimeric luciferase and three auxiliary proteins
The expression of uxC, luxD and luxE gives E. coli the ability to emit photons.

Because luxC, luxD and luxE are included, the luciferase expressing cells need not be given the aliphatic aldehyde substrate luciferin. Since it would be difficult to provide a substrate for eukaryotic luciferase in a biological system as described herein, X. Use the entire lux operon of luminescens. This operon also codes for an enzyme to biosynthesize the aliphatic aldehyde substrate.

X. which is an α-β heterodimer mixed function oxidase. luminescens
Luciferase catalyzes the oxidation of reduced flavins and long chain aldehydes to oxidized flavins and corresponding long chain fatty acids. For the production and regeneration of aldehydes from fatty acids,
A fatty acid reductase complex is required, and NAD (P) H: flavin oxidoreductase supplies reduced flavin.

X. The optimal bioluminescence condition for E. coli expressing the lux gene of luminescens is 37 ° C. (Szittner and Meichen, 19
90, J.A. Biol. Chem. 265: 16581-16687, Xi et al., 1
991, J.M. Bact. 173: 1399-1405). In contrast, luciferases derived from eukaryotic and other prokaryotic organisms typically have lower optimum temperature conditions (Campbell, 1988, Chemiluminescence).
e. Principles and Applications in Bio
logy and Medicine (England Hor, Chichester, UK)
wood and VCH publishers)). Therefore, X. The luciferase from luminescens is suitable for use as a marker for in vivo studies.

All X. The plasmid pGSL1 (F), which contains the luminescens lux operon and confers ampicillin and carbenicillin resistance to Salmonella
The above three strains are transformed by electroporation using rackman et al., 1990). This X. The luminescens lux operon is l
Contains uxA, luxB, luxC, luxD and luxE genes (Fr
ackman et al., 1990). luxA and luxB encode two subunits of heterodimeric luciferase, luxC and luxD encode luciferase substrate biosynthetic enzymes, and luxE is a regulatory gene. In contrast to supplying luciferin exogenously to cells in culture or treating an animal with a substrate, the inclusion of a substrate biosynthetic gene is a convenient means of providing a substrate for luciferase.

(B. Characterization of transformed Salmonella in vitro)
(1. Adhesion and penetration)
The adherence and invasiveness of three Salmonella strains containing the lux plasmid were
inlay and Falkow, 1989, Mol. Microbiol. 3:
Compared to each other in culture and to their non-luminescent parent strains by a general invasion assay (detailed in Example 2) as described in 1833-1841.

In this assay, after culturing with epithelial cell lines and peritoneal macrophages,
Quantify adherent and intracellular bacteria. Adherent bacteria and intracellular bacteria are detected and quantified by both the release of photons from living cells and the colony forming unit after lysis and culturing the cell lysate on a carbenicillin-containing plate.

Some results of this assay are shown in FIGS. 2A-2E and discussed in Example 8. lu
The phenotype of the three strains transformed with the x expression plasmid does not change significantly compared to the parental Salmonella strain. In addition, a good correlation is observed between the intensity of bioluminescence from HEp-2 cells and macrophages and CFU. These results indicate that luminescence as an indicator of intracellular bacteria is a rapid method for assaying the invasiveness of bacteria in culture.

The adhesion of BJ66 to HEp-2 was low compared to SL1344,
The adhesion of these two strains in primary cultures of mouse peritoneal macrophages was comparable.

(2. Light emission)
To examine the oxygen demand of the system, as detailed in Example 3, a 10-fold serial dilution of bacteria is placed in a glass capillary and imaged.

FIG. 3 shows an image created in one such experiment. Luminescence is detected only at the air-liquid interface (0 in 51), even in tubes containing few bacteria in air-saturated medium.
. 1 ml of air saturated buffer will give a final O ₂ concentration of 5 nM).

From these results it is clear that oxygen is probably the limiting factor for luminescence.

(3. Light transmission through animal tissue)
To determine the extent to which light penetrates the animal tissue, the light emitted from the luminescent Salmonella and transmitted through the tissue is used with a scintillation counter with the high-speed simultaneous detector turned off to detect single photons. Quantify. Since the background due to dark current of this type of photomultiplier is significant, this assay is limited to samples with relatively strong photon emission.

Four types of tissue with different clarity are compared in this way: chick breast muscle, chick breast skin, lamb kidney, and kidney medulla obtained from lamb kidney.
The number of photons that can be detected through the tissue is about 10 times less than controls that do not pass through the tissue.

(4. In vivo characterization of lux Salmonella)
(A. Oral administration)
Oral inoculation is a natural route of Salmonella infection for mice and humans, resulting in more delayed disease progression. To study the progression of Salmonella infection by this route of inoculation, two strains of mice are infected with three strains of Salmonella. The results obtained using resistant animals are discussed below under the heading "Infecting resistant mice".

Balb / c mice were treated with pathogenic SL1344lux as described in Example 5.
Non-invasive BL66lux and low pathogenic LB5000lux Salmonella are orally infected. The progress of the infection is followed over 8 days by external imaging ("Materials and methods" section).

Typical images are shown in FIGS. At 24 hours post inoculation (pi), the bioluminescent signal is localized to a single lesion in all infected animals (FIGS. 5A, 5C and 5E). By 7 days after inoculation, bioluminescence disappears in all animals infected with low pathogenic LB5000lux (FIG. 5B). In contrast, pathogenic SL1344l
For animals infected with ux, the time at which spread begins varies considerably from animal to animal, but often shows severe infection that spreads to the majority of the abdominal cavity (FIG. 5F). BJ66lu
Infection with x typically persists and remains localized at a single site (FIG. 5D).

(B. Intraperitoneal inoculation)
Oxidation of luciferin followed by luminescence at the replication site of Salmonella (Campbe
ll, 1988, Chemiluminescence. Principles
and Applications in Biology and Med
To assess whether there is enough O _{2 in} icine (Chichester, UK: Ellis Horwood and VCH Publishers), light emission from tissues of breathing animals is measured. Luminous SL1344lux and LB5000lu
x is inoculated intraperitoneally into two groups of Balb / c mice. At 32 hours after inoculation (pi), the transmitted photons are imaged (FIG. 6).

In mice infected with SL1344lux (left side of the figure), the transmitted photons are visible on a large surface and various intensities of focus are visible. These images show disseminated infection and are consistent with extensive colonization of the viscera (possibly including the liver and mesenteric lymph nodes). In contrast, the distribution of transmitted photons from animals infected with LB5000lux is very limited, indicating limited infection.

LB5000lux infected mice remained healthy for several weeks after inoculation,
SL1344lux-infected mice became nearly moribund 4 days after inoculation and were euthanized.

These experiments indicate that O ₂ levels in blood or tissue are sufficient for the bioluminescence of lux luciferase expressed from Salmonella. Also,
These experiments show that the pathogenic strain S compared to the laboratory strain LB5000 with reduced pathogenicity.
This is consistent with the strong penetration of L1344.

(C. Infection of resistant mice)
Mice where the Ity locus is heterozygous (Ity ^{r / s} ) are resistant to systemic infections by Salmonella typhimurium (Plant and Glynn,
1976, J.A. Infect. Dis. 133: 72-78). Bcg (Gro
s et al., 1981, J. Am. Immunol. 127: 2417-2421) or L
sh (Bradley, 1977, Clin. and Exper. Immun
ol. 30: 130-140), this locus is a mycobacteria (Myc
obecium lepraemurium; Forget et al., 1981
, Infect. Immunol. 32: 42-47), Mycobacterium bovis (M. bo
vis; Skamen et al., 1984, Immunogenet. 19: 117
-120, Skamene and Pietrangeli, 1991, Natu
re 297: 506-509) and M. intracellulare (M. intracellulare)
are; Goto et al., 1989, Immunogenetics 30: 218.
Regulate the pathogenesis process of certain intracellular pathogens such as -221). Similar genetic control of resistance and sensitivity to intracellular pathogens appears to exist in humans (
Mycobacterium tuberculosis (M. tuberculosis; Stead, 1992, Ann)
als of Intern. Med. 116: 937-941, Stead et al., 1990, New Eng. J. et al. Med. 322: 422-427) and M. leprae).

The Ity locus is present in mouse chromosome 1 in two allelic forms, Ity ^r (tolerant, dominant) and Ity ^s (sensitive, recessive). The gene encoded at the Ity locus affects the ability of macrophages to destroy internalized pathogens (Blackwell et al., 1991, Immunol. Lett. 30: 2).
41-248 (1991); Skamen et al., 1984, I.
mmunogenet. 19: 117-120 and Skameene and Piet
See also rangeli, 1991, Nature 297: 506-509), which appears to affect the downstream function of pathogen transport to other sites within the infected host where it is mediated by macrophages. Balb / c mice are It
y ^{s / s} , and 129 mice are Ity ^{r / r} . In the experiments detailed here, heterozygous Balb / cX129 mice (Ity ^{r / s} ) are used.

As detailed in Example 7, resistant 129XBalb / c (Ity ^{r / s} ) surviving mice are infected by intragastric inoculation of 1 × 10 ⁷ SL1344lux Salmonella. The mice are imaged daily for 8 days (8 dpi) after injection.

Results are shown in FIG. 7A (first day) and FIG. 7B (eighth day). Luminescence detected by external imaging was evident at 24 hours after inoculation and appeared to be localized to a single site in all animals. The luminescent signal is always present throughout the test period (up to 8 days after inoculation). The intensity of light emission and the location of the light source vary somewhat over time within a single mouse and can also vary from mouse to mouse. In any infected animal, the luminescent tissue is caecum (see below), and the variability in location is probably due to the fact that the internal organs of the rodent are not strongly fixed in place (possibly also the intensity variability). Let's go.

The apparent limited infection observed in these animals supports the interpretation that this Ity restriction blocks macrophage trafficking. However, the persistence of this infection for 10 days suggests that there is adherence to the intestinal mucosa and that the detachment of bacteria continues in the faeces of these animals as evidenced by the luminescent faeces. is doing. These results indicate that the luminescent phenotype of Salmonella in vivo can be maintained for 8 days in Ity-restricted animals and can be localized after oral inoculation.

(D. Internal imaging after oral inoculation)
Infected mice are imaged after abdominal wall incision to further localize the luminescent signal in the abdominal cavity (FIG. 8). In any animal infected by the oral route, the main disease symptom is dilation of the cecum (FIGS. 8A-C). The “external” image (FIG. 8A) shows localized luminescence, which can be seen in the image after the abdominal wall incision (FIG. 8B) hitting the cecum.

Injecting air into the intestine confirms the presence of bacteria in other areas of the digestive tract. Bacteria in the colon and rectum appear to express luciferase, but luminescence from those sites appears to be limited by low oxygen concentrations.

The images obtained in the oral inoculation test show that the luminescence signal is 2 days after inoculation and 7 days after inoculation
Except for mice infected with LB5000lux, it has been shown to be almost completely localized in the cecum of each animal (Popesko et al., 1990, A Color.
Altas of Anatomy of Small Laboratory
Animals Vol. Two: Rat Mouse Hamster (London, England)). Luminescence is also visible in several colons. By 7 days after inoculation, no luminescence is observed in LB5000lux-infected animals. CFU present in the organs of these mice is measured 2 and 5 days after inoculation.

In animals infected with the invasive strain SL1344lux intragastrically, luminescence in the cecum appears early, followed by systemic infection. In contrast, non-intrusive BJ66l
Infection with the ux strain results in persistent luminescence from the cecum, which is the duration of the entire study
Some animals are maintained over a period of days. By 8 days after inoculation, luminescence is detected on the majority of the abdominal surface, which resembles the distribution of photons after intraperitoneal inoculation in SL1344lux infected mice.

As expected, infection with SL1344lux appears to be systemic, with increasing surface area emitting photons and progressively increasing photons emitted therefrom.
With any strain infection, luminescence is localized on the abdomen and little luminescence from outside this region appears to be detected. A large number of transmitted photons are localized as a single focal point on the abdomen, suggesting that most of the replication is in the area surrounding the intestine, even if the infection is systemic.

Localization of luminescence on the cecum not only indicates that there is a large amount of organisms in this area of the intestine, but also that Salmonella binds to the cells of its mucosa so that there is enough oxygen to luminescence. It also suggests. Photon emission from luciferase is oxygen dependent and the expected oxygen level in the cecal or small intestinal lumen is lower than that required for luminescence. The luciferase reaction is not expected to function in the gut unless the bacterium obtains oxygen from cells of the intestinal epithelium.

Thus, systemic infection may be associated with an invasive phenotype and not simply attachment to intestinal epithelial cells. These experiments imply that the cecum has some role in the carrier state of this pathogenic process or as a seeding site.

Monitoring the progression of infection to different organizations can significantly deepen understanding of these stages in the pathogenesis process and enable screening of compounds that are effective at controlling the pathogen at the selected stage. Let's go.

(E. Internal imaging after intraperitoneal inoculation)
Mice infected with SL1344lux intraperitoneally are imaged before and after abdominal wall incision (Example 9). The result is shown in FIG. The image shows light emission with multiple transmitted photon focal points in the majority of the abdomen. The cecum does not appear to contain luminescent Salmonella. The results obtained from these experiments indicate that all Salmonella strains have O ₂ sufficient to emit light in the early phase of infection. However, since systemic infection at a later time point is only seen in SL1344lux-infected mice, it appears that Salmonella mucosal cell entry and subsequent systemic infection is limited to strains with an invasive phenotype .

(F. Effect of ciprofloxacin on Salmonella infection)
To demonstrate that non-invasive imaging is useful for tracking the response of an infection to a drug, the experiment detailed in Example 10 is conducted. SL1344lux on the mouse
100 ml of the effective antibiotic ciprofloxacin for Salmonella infection
Treat with g. At selected times after treatment, the mice are imaged and the extent of infection is quantified by measuring photon emission. The photon emission in the treated mice is compared to the value before the start of treatment and the value from control mice that were infected but not treated. The results obtained in one such experiment are shown in FIGS. 10A-E and discussed in Example 10.
Infections in mice treated with antibiotics are always low during the treatment period compared to the pathogen levels at 0 hours in treated mice and in control mice.

(G. Effect of carbenicillin selection)
Duclzeau et al., 1970, Zeut. Bakt. 5313: 533-
548 showed that treatment of animals with antibiotics promotes cecal colonization by Salmonella. Mice in this experiment are maintained with antibiotic treatment, intramuscular injection of carbenicillin, to select Amp ^r Salmonella containing luciferase clones. Although this treatment may change the course of gastrointestinal infections, the observation that Salmonella can bind to the cells lining the cecum indicates that oxygen can be used for luminescence. This observation is notable because the cecal lumen is generally considered an anaerobic environment.

(VIII. Application)
Bioluminescent technology is widely applicable to various host-pathogenic systems and also allows for spatiotemporal evaluation of other biological events, such as tumor progression and gene expression in living mammals right. It can also be applied to drug development and screening. The widespread use of in vivo imaging of pathogens could reduce the number and time of animals required for experiments on pathogenic and / or real-time test antibacterial agents. Also, luminescent bacteria are used for environmental analysis, but bioluminescent organisms may be useful as biosensors in living animals. For example, Korpela
Have limited oxygen supply within the lumen of the gastrointestinal tract, so that oxygen can be directly accessible to salmonella (possibly from epithelial cells or other cell types)
This indicates that bioluminescence is limited. Korpela et al., 1989, J. MoI. B
iolum. Chemilum. 4: 551-554. This oxygen demand could be used as an indicator of intimate cell-cell interactions or as a biosensor to examine oxygen concentrations at various sites in a living animal. Less than,
Several exemplary applications of this technology are described for purposes of illustration and are not intended to limit the invention in any way.

(A. Measurement of oxygen level)
The need for oxygen for luciferase luminescence, as demonstrated in the experiments summarized above, indicates that the present invention can be used as a method for measuring the spatial gradient of oxygen concentration in a subject. Luminescent bacteria have been used to measure oxygen levels in the 10-1 mM range. According to these studies, 0.1 nM is expected to be the lower detection limit (Campbell, 1988, Chemilumines).
ence. Princes and Applications in
Biology and Medicine (Chichester, UK: Ellis)
Horwood and VCH publishers)). The imaging methods described herein can be used to examine oxygen levels at various sites within a living animal. For example, microorganisms that have been engineered to emit light in O ₂ or in a Ca ²⁺ dependent manner,
Just as luminescent bacteria are used for environmental analysis (Guzzo et al., 1992,
Tox. Lett. 64/65: 687-693; Korpela et al., 1989
, J .; Biolum. Chemilum. 4: 551-554; Jassim et al., 1990, J. MoI. Biolum. Chemilum. 5: 115-122) could be used as a biosensor within a subject. The dynamic range of luminescence with respect to O ₂ concentration is much wider than O ₂ probes, reaching lower O ₂ concentrations (Campbell, 1988, Chemiluminescence).
. Principles and Applications in Biol
ogy and Medicine (Chichester, England: Ellis Horw
ood and VCH publishers)). Furthermore, the light emission proportional to the O ₂ concentration is 30 n
Linear over the range from M to 8 mM, 9 mM for ½ maximum emission
O ₂ is required.

(B. Localization of tumor cells)
Tumor growth and metastatic spread within a subject can be monitored using the methods and compositions of the invention. Especially when the individual is diagnosed as having a primary tumor, the LEC for the cells of the tumor is used to clarify the boundary of the tumor, and the cells have moved away from the primary tumor mass and settled You can decide whether or not.

For example, an LEC such as a liposome containing an antibody against a tumor antigen and added with LGM is administered to a subject, bound to a tumor cell in the subject, imaged, and the photon emission region is defined as a tumor cell region. Can be correlated.

As a related aspect, tumor growth, progression and metastasis can be monitored over time within a subject by creating images using tumor localized LEC as described above at selected time intervals. . Such monitoring would be useful for recording anti-tumor therapy results or as part of a screening for therapeutic compounds presumed to be useful for inhibiting tumor growth or metastasis.

Tumor cells can also be transformed with a luciferase construct under the control of a constitutively active promoter and used to induce a luminescent tumor in an animal model as described above. Such animal models can be used to evaluate the effects of candidate anti-tumor compounds.

(C. Localization of inflammation)
By using the composition and method of the present invention in the same manner as described above, the site of inflammation can be identified, inflammation can be monitored over time, and / or anti-inflammatory compound candidate can be screened. . Molecules useful for directing to sites of inflammation include the ELAN protein family that binds selectins. ELAN molecules can be incorporated as targeting components on the objects of the present invention and used to target inflammatory sites.

Moreover, an animal model for studying anti-inflammatory substance candidates can be created by creating an animal genetically modified to have luciferase under the control of the E-selectin promoter. Since E-selectin is expressed at the site of inflammation,
Transgenic cells at the site of inflammation will express luciferase.

This system can be used for screening for anti-inflammatory substances. Evaluate the effects of candidate anti-inflammatory compounds by administering inflammatory stimuli to control and experimental animals and comparing the effects of candidate anti-inflammatory compounds on luminescence induced in treated animals with control animals be able to.

(D. Localization of infection)
As illustrated in the above experiments conducted to support the present invention, LGC
It can be used effectively to track the course of infection of a subject by a pathogen. In the experiments detailed herein, LGC is a pathogenic cell (Salmonella) that has been transformed to express luciferase. Such a system is ideal for studying infection and subsequent spread of infection in animal models of human disease. According to such a system,
Infectious disease progression can be monitored using the site of infection and the site of disease progression rather than traditional systemic symptoms such as fever and swelling for pathogenesis studies.

Using external imaging to monitor the efficacy of anti-infectives allows temporal and spatial assessments in individual living animals, thereby allowing pathogens and / or
Or the number of animals required for experiments on the test anti-infectives can be reduced.

The following examples illustrate the invention, but are not intended to limit the invention in any way.

(Materials and methods)
(A. Cell)
Salmonella strains SL1344 and LB5000 are A. D. Stocker (
Stanford University; Hoiseth and Stocker, 1981, Nat
ure 291: 238-239). Salmonella strain BJ66 is
D. Obtained from Jones (Stanford University).

HEp-2 cells are the American Type Culture Colle.
ction (ATCC; Rockville Park Lane Drive, Maryland 12)
301; accession number CCL-23).

Mouse peritoneal macrophages are located 7m in the peritoneal cavity of euthanized Balb / c mice.
obtained by washing with 1 growth medium (Maximow and Bloom,
1931, Textbook of History, Saunders,
Philadelphia).

(B. Static culture)
Hypoxia (static) is obtained by inoculating 3 ml of LB broth containing 100 mg / ml carbenicillin with 6 μl of the bacterial suspension obtained from stationary phase culture and growing the bacteria overnight at 37 ° C. in a 7 ml stationary culture tube. C) A culture was prepared.

(C. mouse)
Balb / c (Ity ^{s / s} ) mice were used at the oncology department at Stanford University (t
he Department of Oncology). 129
XBalb / c (Ityr ^{/ s} ) mice are the Stanford Transgenic Animal F
(acity; Stamford, CA). All animals have the same photoperiod, feeding method and temperature conditions, and the Stanford University Research Animal Facility.
Animal Facility; Stamford, California).

Anesthesia was performed by injecting 33 μg / kg body weight of Nembutal intraperitoneally into the animals.

Euthanasia was performed by CO ₂ asphyxiation or cervical dislocation according to protocols recommended by the Stanford Research Animal Facility. Cervical dislocation was used when physiological changes due to suffocation might affect experimental results.

Mice infected with lux-transformed Salmonella are injected intramuscularly (im) with carbenicillin (body weight) in order to keep the luminescent Salmonella under selective pressure such that the Amp ^r plasmid containing the lux operon is retained. 125m per kg
g) was applied daily.

(D. Imaging)
A charge-coupled device (CCD) camera with a two-stage microchannel sensitizing head (model C2400-40, Hamamatsu)
And fixed in a light-tight box with a door. The camera was connected to an “ARGUS50” image processing device (Hamamatsu) via a cable leading out of the box.

The ICCD system described above can detect a single photon once it reaches the threshold of 10-30 photons. The signal-to-noise ratio of this system is 2 depending on the signal strength:
It ranged from 1 to 1 × 10 ⁴ : 1.

The grayscale image was obtained by opening the door of the light box in a faint room light and integrating for 8 to 64 frames. The gain of the grayscale image was set to optimize the image (typically 3000 volts on a scale of 0 to 10,000 volts).

Bioluminescence data was obtained in the absence of external irradiation. The exposure settings were as follows: the black level was automatically set by the camera / image processor, the gain was automatically set by the multiplier controller, and the F-stop was set to 2.8. A 60 mm “AF Nikkor” macro lens was used (Nikon, Melville, NY).

Bioluminescent images were generated by integrating photons for a selected time (typically 5 minutes). Data is represented for each animal with a minimum bit range setting of 0 to 3 bits per pixel. For images of other objects where the bioluminescence signal resolution could not be in the 0-3 bit range (ie 24 well plates), the range should be set to a setting that can localize the bioluminescence signal (typically 1-7) Increased.
If no additional information was obtained after 5 minutes of imaging, the object was imaged in a shorter time.

External imaging refers to non-invasive imaging of animals. Internal imaging refers to imaging after a partial incision (typically an abdominal wall incision) in an animal. Internal imaging is performed on selected animals to identify the source of photon emission localized by external imaging.

The bioluminescence image data is represented as a pseudo color luminescence image representing the intensity of the detected photon. Typically, six intensity levels from blue (low intensity) to red (high intensity) are used.

In order to create the figures shown here, a grayscale image and a bioluminescent image were superimposed using an image processor to create a composite image that gave a spatially compliant coordinate system.

The composite image was displayed on an RGB CRT (red, green, blue; CRT) monitor, and the monitor was photographed to make a hard copy. The hard copy was also made by storing the image processing device image as a digital file, transferring the file to a computer, and printing it with a color printer connected to the computer. Alternatively, a hard copy may be made by printing the video signal directly using a video printer.

Example 1
(Transformation of Salmonella with pCGLS1lux plasmid)
Salmonella strains SL1344, BJ66 and LB5000 were transformed into Xenorha
The pUC18 vector pCGLS1 encoding the lux operon derived from bdus luminescens (Fraccman et al., 1990) was transformed.

(A. pCGLS1 plasmid)
A schematic of the pCGLS1 plasmid is shown in FIGS. 1A, 1B and 1C. This plasmid is a lux derived from the soil fungus Xenorhabdus luminescens.
˜11 kb region encoding the gene (FIG. 1A; Frackman et al., 1990)
PUC18 (FIG. 1C; Clontech, Palo Alto, Calif.)
Was constructed by cloning into the Bam HI site (FIG. 1B). The construction of this vector is described in the report by Frackman et al. (1990).

The restriction enzyme sites in FIG. 1A are described as follows: Bs, Bst EI
I; C, Cla I; E, Eco RI; H, Hind III; M, Mlu
I; S, Sca I; X, Xba I; B / Sa, Bam HI and Sau
3A junction. The sequence containing the multiple cloning site (MCS) is set forth in FIG. 1B and its Bam HI site is shown in bold.

A schematic of the pUC18 vector without the insert is shown in FIG. 1C. Marked elements include the ampicillin resistance gene (Ap), the lac Z gene (lac Z), and the origin of replication (Ori) of E. coli. The size of the unmodified pUC18 vector is about 2.7 kb.

(B. Transformation of Salmonella)
Electrocompetent cells of Salmonella strains S1344, BJ66 and LB5000 were prepared by a general method (Sambrook et al., 1989, Molecular.
r Cloning: A Laboratory Manual, Cold S
spring Harbor Laboratory Press, Vol. 2) and stored at −80 ° C. until just before use. Electroporation was performed as follows. 1 μl of plasmid (0.2 μg / ml) was added to 40 μl ice-cold electrocompetent cells suspended in 10% glycerol. 1 of the suspension
Mix gently for 1 minute and place in an electroporation cuvette with 1 mm gap,
Bio-Rad Gene-Pluser (Bio-Rad Laborato
Ries, Hercules, Calif.). The settings were 2.5 kilovolts, 400 ohms and 25 microfarads.

After stirring for 1 hour at 37 ° C. in Luria-Bertini (LB) broth, 100
Cells were seeded on (LB) agar containing μg / ml carbenicillin and grown overnight.

To maximize the bioluminescence of labeled Salmonella, the lux operon was maintained on a high copy number plasmid so that it did not integrate as a single copy gene. However, the plasmids are r r such as SL1344 and BJ66 used in this study.
The ecA strain is particularly modified by bacterial cells. The recA locus is lux
It encodes a recombinase that can delete a region of the plasmid containing the operon and β-lactamase. Therefore, Salmonella recovered from cultured cells were plated in both the presence and absence of carbenicillin and imaged to determine the frequency with which bioluminescence was lost. All colonies recovered from gentamicin-treated and lysed HEp-2 cells and macrophages were ampicillin resistant (Amp ^r ) and bioluminescent. Thus, the lux gene did not appear to be lost during co-culture with mammalian cells.

Colonies were examined for luminescence by visual recognition in a dark room. Five transformants were identified as having high levels of luminescence. Three of these (SL1344, BJ6
6 and 1 each from LB5000 strains) were selected for subsequent experiments.
They are SL1344lux, BJ66lux and LB5000lu respectively.
It was named x.

(Example 2)
(Invasion ability of normal and transformed Salmonella)
6 strains of Salmonella (SL1344lux, LB5000lux, BJ66lu
x, SL1344, LB5000 and BJ66) were measured in two bacterial attachment and invasion assays. The colony forming unit (CFU) assay is
Basically the method already described (Finlay and Falkow, 1989
Mol. Microbiol. 3: 1833-1841) (Lee et al.,
1990, PNAS 87: 4304-4308). The bioluminescence assay was basically performed in the same manner as the CFU assay except that the number of cells was quantified using bioluminescence instead of CFU.

Briefly, RPMI with HE-p cells and primary mouse peritoneal macrophages added to a 24-well tissue culture dish with 20 mM glutamine (Gibco / BRL) and 5% fetal calf serum (Hyclone, Logan, Utah). Gibco
/ BRL, Grand Island, NY) at a concentration of 1 × 10 ⁵ cells / well. 24 hours after seeding the cells (HEp-2) or several days later (
Macrophages) with bacteria from static culture (see “Materials and Methods” above),
1 × 10 ⁶ per well (multiplicity of infection (moi) 10) or 1 × 10
Inoculate at a density of ⁷ (moi = 100, right column in FIGS. 2B-E), Beck
Using a man clinical centrifuge (Beckman Instruments, Columbia, MD), the mixture was centrifuged on a cell monolayer at 1000 rpm (185 × g) for 5 minutes. RPMI medium (Gibco / BRL) with or without gentamicin (100 mg / ml) (invasion assay) or without (adhesion assay)
Replaced with. The co-culture was cultured at 35 ° C. in 5% CO ₂ for a total of 3.5 hours.

Gentamicin in the culture medium kills bacteria that have not been internalized by Hep-2 cells, including those attached to the surface of HEp-2 cells. Therefore,
The signal in the attachment assay (without gentamicin) represents both adherent and internalized bacteria, whereas the signal in the invasion assay (with gentamicin) represents only the internalized bacteria.

Attachment and invasion were assayed by imaging luminescent bacterial cells at 3 time points (1.5 hours, 3.0 hours and 3.5 hours after inoculation). Prior to imaging at the first time point, cell monolayers were washed with phosphate buffered saline (to remove unattached bacteria).
PBS) was added 3 times and fresh RPMI was added. Luminescence was recorded with an exposure time of 30 seconds. Images at the second and third time points were also obtained using the same exposure time, but the cells were not washed first.

FIG. 2A shows the data recorded at the last time point as a pseudo-color emission image superimposed on the grayscale image of the culture dish well. The cell type, Salmonella strain and gentamicin usage are shown in the figure. This data is also summarized in the graphs of FIGS. 2B and 2D as the relative intensity of the photon count.

After imaging at 3.5 hours, tissue culture cells were washed 3 times with PBS and lysed with 0.2% “Triton X-100” in PBS. Adherent bacteria and / or intracellular bacteria released by lysis were plated on LB agar plates or LB-carbenicillin agar plates and cultured at 35 ° C. for 18 hours. The number of bacteria released from each well was expressed as colony forming units (CFU, Finlay and Falkow, 1989
Mol. Microbiol. 3: 1833-1841; Lee et al., 1990.
, PNAS 87: 4304-4309).
These data are expressed as total bacterial colonies per ml recovered from co-cultures after 3.5 hours of culture with or without gentamicin and are summarized in the graphs of FIGS. 2C and 2E.

The data obtained from both the bioluminescence assay and the CFU assay are (i) lu
the Salmonella transformed with the x gene has infectivity similar to that of the parent line, (
ii) It is shown that luminescence detection and CFU determination give equivalent estimates for the invasion power of the two Salmonella strains in HEp-2 cells and macrophages. CFU
The ratio of bioluminescence to is lower in macrophage cultures, probably because Salmonella enters the subcellular compartment of macrophages.

(Example 3)
(In vitro luminescence of transformed Salmonella)
Four 10-fold serial dilutions of LB5000lux Salmonella (from 10 ⁶ cells to 10 ³ cells per ml) were added to four 100 μl glass capillaries (Be
Cton Dickinson's Cary-Adams Division, Parsippany, NJ). The bacterial suspension formed liquid columns with air pockets at both ends in the capillary. One end of each capillary tube is clit sealed
(sea; Clay-Adams). The medium used to make the dilution was saturated with O ₂ by exposure to air.

The capillary was wrapped in a clear plastic wrap and imaged for 30 seconds as described above to measure luminescence. A typical image is shown in FIG. 3A. Four capillaries are shown. These include (from top to bottom) 10 ⁶ , 10 ⁵ , 10 ⁴ and 10 ³ Salmonella cells / ml (10 ⁴ , 10 ³ , 10 ² and 10 cells / tube). Luminescence could be detected even in a suspension containing only 10 ⁴ cells / ml (100 cells). However, its emission is limited to the air / liquid interface, suggesting that this luminescent reaction requires a relatively high level of oxygen. Many of the cells are probably in the liquid column and air /
Since this would not be at the liquid interface, this data indicates that the luminescence in the capillary shown in FIG.
It suggests that it results from significantly fewer cells than the total number of cells in each tube.

Example 4
(In vitro detection of luminescence penetrating animal tissues)
A micro-test tube composed of a 3.5 mm inner diameter glass capillary containing a serial dilution of LB5000lux Salmonella was prepared essentially as described in Example 3 above. However, in this example, the bacterial suspension was brought into contact with the sealed end of the capillary and only the top was exposed to air. The tube was placed in a translucent plastic scintillation vial and surrounded by one of the following animal tissues: chick pectoral muscle,
Chick skin, lamb kidney or lamb kidney medulla. Both organizations were obtained from the meat department of a local supermarket (Safeway, Mountain View, Calif.).

A diagram of a vial containing a capillary tube surrounded by tissue is shown in FIG. Vial 1 is approximately 1.4 cm in diameter and includes a lid 2. The vial is covered with an opaque material (ie black tape) along its upper portion 3. Apply animal tissue 4 from the bottom of the vial to the opaque coating 3
Place in a vial so that it is just above the bottom edge of. Plug the bottom of the micro test tube 5 into the stopper 7 (
That is, seal with a crytoseal stopper and place it in the center of the vial so that the stoppered end contacts or is very close to the bottom of the vial. The bacterial suspension 6 reaches about 1 cm above the bottom of the capillary.

Liquid scintillation counter (model 1219 Rackbeta, LKB / W) with high-speed simultaneous discriminator disabled for photons emitted from vials with and without tissue and vials with and without bacteria.
Allac, Gaithersburg, Maryland).

Tissue-free controls were assayed by placing the bacterial suspension directly into scintillation vials. All experiments were performed in triplicate.

For each experiment, rotate the vial 90 ° C for each count,
By counting three times, the influence of possible thickness mismatch of the organization was verified. No significant difference was detected.

  The results are summarized in Table 1 below.

The signal for 1 × 10 ³ LB5000lux in the kidney tissue was at or near the background level with a photomultiplier tube (PMT) in the scintillation counter. The background in this type of detection is due to the dark current of the PMT, and this test is limited to the analysis of fairly strong signals.

Bioluminescence from approximately 1 × 10 ⁷ LB5000lux could be detected through 0.5 cm avian muscle, skin, sheep kidney medulla and sheep kidney. These results indicate that bioluminescence from labeled Salmonella can be detected through animal tissues of varying opacity. Since oxygen appears to be limited within the capillary (as shown in FIG. 3A), it is believed that fewer bioluminescent Salmonella than can be detected through the tissue than the number indicated in this assay.

(Example 5)
(In vivo detection of bioluminescent Salmonella)
To assess the availability of oxygen to infected Salmonella, wild type SL13
44 lux was inoculated intraperitoneally (ip) in BALB / c mice. 2 of inoculation
Four hours later, in anesthetized rats, photons emitted by bacteria and transmitted through the abdominal wall were detected from the outside with a multiplying CCD camera and localized (FIG. 3B). Systemic Salmonella infection may involve lymph node, spleen, and liver colonization.
Abdominal images of mice infected by intraperitoneal inoculation with wild-type SL1344 lux showed that transmitted photons had various intensities of focus on most of the abdominal surface (FIG. 3B). These results are consistent with extensive colonization of the viscera, presumably including the liver and mesenteric lymph nodes, and available oxygen levels can be used to detect luminescence from labeled pathogens externally in some tissues. It shows that it can be sufficient.

(Example 6)
(Detection of lux Salmonella orally administered to Balb / c mice)
Pathogenic SL1344lux, non-invasive BJ66lux and low pathogenic LB50
Balb / c mice were infected by oral administration of 50 μl of ⁷ suspensions of 00lux Salmonella 1 × 10 ⁷ . Those mice that were 4-6 weeks old at the time of infection were imaged daily with a 5 minute integration time (photon emission was measured for 5 minutes). Prior to imaging, mice were anesthetized with 33 μg / kg body weight Nembutal.

Typical images are shown in FIGS. At 24 hours post-inoculation (pi), the bioluminescent signal was localized to a single focal point in all infected animals (FIGS. 5A, 5C and 5E). Bioluminescence disappeared by 7 days after inoculation in all animals infected with low pathogenic LB5000lux (FIG. 5B). In BALB / c mice infected with wild-type SL1344 lux, bioluminescence was always detected during the study period, and on the 8th day, multiple transmitted photon foci were observed. In these animals, the infection often spread to most of the abdominal cavity (FIG. 5F). In one-third of these animals, transmitted photons were found in most of the abdominal area on day 8, which was similar to the distribution of photons after intraperitoneal inoculation (see FIGS. 3B and 5F). Although the prevalence of infection with BJ66 lux was rather variable, typically the infection persisted and remained localized at the initial site (FIG. 5D).
).

When resistant BALB / cX129 mice were infected with wild type SL1344 lux, the bioluminescence signal was always localized and persistent in the group of 10 mice during the test period. This result was in contrast to the disseminated bioluminescence observed in susceptible mice infected with SL1344 lux (lyt ^{r / s} ) (see Example 8 and FIGS. 7A and 7B), but invasive. It was similar to persistent infection of susceptible BALB / c mice with low BJ66 lux. As a control, Salmonella obtained from persistently infected resistant BALB / cX129 mice was cultured and 80-90% of the colonies recovered after 8 days were Amp ^r . 9 of these
More than 0% was bioluminescent, suggesting that the observed differences were not due to a significant decrease in lux plasmids, but rather because the pathogenicity of the bacterial strains was truly different.

(Example 7)
(Detection of infection after intraperitoneal inoculation of Salmonella pathogenic and low pathogenic strains)
Balb / c mice were made virulent (SL1) by inoculating intraperitoneally (ip) without simultaneous air infusion of 1 × 10 ⁷ bacterial cells in 100 μl suspension.
344 lux) or low pathogenicity (LB5000 lux) Salmonella.

At 32 hours after inoculation (pi), mice were anesthetized and imaged as described above. The result is shown in FIG. Two mice on the left side of the figure (pathogenic SL1344
Widespread infection is evident in lux infection). In contrast, low pathogenic LB5
In the right mouse injected with the 000lux strain, little if any luminescence is detected.

(Example 8)
(Detection of systemic infection in resistant mice after oral inoculation of Salmonella)
Resistant 129XBalb / c (Ity ^{r / s} ) surviving mice were treated with 1X10 ⁷ S
Infections were obtained by intragastric inoculation of L1344lux Salmonella. Bacteria were given through an intragastric feeding tube under anesthesia. Animals were imaged daily for 8 days after injection (8 dpi).

The results are shown in FIGS. 7A and 7B. A set of 3 mice were infected and imaged daily for 8 days. A typical image of day 1 (FIG. 7A) and a typical image of day 8 (FIG. 7B) are shown. These data indicate that mice resistant to systemic Salmonella infection have a chronic infection localized in the cecum, but the infection does not spread intraperitoneally.

Example 9
(Image taken after abdominal wall incision after oral inoculation of Salmonella)
An abdominal wall incision was made after oral inoculation of Salmonella to pinpoint the location of the luminescent signal in the abdominal cavity and compare that position with that obtained by noninvasive imaging. Animals were inoculated as described in Example 8. After a selected period (typically 7 days), the mice were anesthetized as described above and imaged externally. A typical image is shown in FIG. 8A. After external imaging, the abdominal cavity was opened and the animal was imaged again as illustrated in FIG. 8B. In some cases, air was injected into the intestinal lumen before and after the cecum (C), and then the third imaging was performed (FIG. 8C). Mice were euthanized immediately after the last imaging.

In all cases where bioluminescence was observed locally in susceptible mice after oral inoculation, photons originate almost exclusively from the cecum, and the precise localization and variation in intensity of localized bioluminescence is This was because the position of the cecum was easy to change. Infection resistance B
The localized bioluminescence observed in ALB / cX129 mice was also localized in the cecum as well. In contrast, such localization was not observed in animals infected intraperitoneally with SL1344 lux (FIG. 3B). In late stages of infection-sensitive mice orally inoculated with wild-type SL1344 lux, bioluminescence was multipolar, but the focus of added luminescence did not become apparent after abdominal wall incision. Low pathogenicity LB5000
In mice infected with lux, as of day 7, no bioluminescence could be detected in any tissue or organ, even after localized removal of the skin and abdominal wall.

No bioluminescence was detected optically in the spleen or bloodstream of any infected animal.
Bioluminescence from the liver was observed only in the late stage of the disease. In addition, bioluminescence from the gastrointestinal tract was limited to the cecum at the beginning of the disease. This pattern may be due to differences in the number of Salmonella in different tissues or lack of available oxygen. The distribution of labeled Salmonella SL1344 lux was examined by quantifying Amp ^r cfu present in crushed organs of orally infected mice. 7th day SL1344 lu
x infectious BALB / c mouse liver, # 038 ^r bacterial colonies obtained from the spleen and lungs, both showed total cfu in the range more than 90% of 1.9X10 ³ ~> 1.0X10 ^5, in vivo There was no detectable photon emission (Table 2). In contrast, bioluminescence from the cecum was detectable, and the tissue contained> 1.0 × 10 ⁸ total cfu. In LB5000 lux infected mice, cf
u was not detected. These results suggest that 1 × 10 ⁶ organisms in the tissue are near the limit of detection at this emission wavelength when using current experimental systems.

Since oxygen is an essential substrate for the luciferase reaction, only Salmonella present in the oxygenated microenvironment should be bioluminescent. Therefore, it can be expected that there is no bioluminescence from Salmonella in the anaerobic environment of the lumen of the gastrointestinal tract,
Bringing the intestinal lumen to air should reveal the presence of bacteria that were previously undetectable due to the lack of oxygen. In support of this view, one animal with bioluminescence detectable only in the cecum excreted feces that rapidly become bioluminescent when exposed to air. Given the fact that there are non-luminous luciferase-expressing bacteria in the intestinal lumen, and a clear depiction of the aerobic and anaerobic zones in this tissue, the presence of additional bacteria when infused with air It was suggested that would become clear. When air was infused into the ileum and colon lumens of another animal showing a similar bioluminescence pattern, photons could be detected near the injection site (FIG. 8). Finally, when the third mouse that exhibited cecal bioluminescence A was killed, the bioluminescence disappeared rapidly. In order to lack clear aerobic and anaerobic zones, air was injected into other tissue sites.

(Example 10)
(Imaging after abdominal wall incision after intraperitoneal injection of Salmonella)
As described in Example 7, 1 × 10 ⁷ Salmonella (SL1344lux
) Was intraperitoneally inoculated Balb / c mice. One such typical image is shown in FIGS. 9A, 9B and 9C.

At 24 hours post injection (pi), the animals were anesthetized and imaged for 5 minutes (Figure 9A).
). The abdominal cavity was opened and the mouse was imaged again for 5 minutes (FIG. 9B). The cecum was pulled to the left and the animal was imaged again for 5 minutes (FIG. 9A).

From the results, it can be seen that the localization of the infected site obtained by the noninvasive imaging method correlates well with the site revealed by opening the abdominal cavity.

Example 11
(Effect of ciprofloxacin on bioluminescence from SL1344lux Salmonella)
In order to demonstrate the usefulness of in vivo imaging, infected animals were treated with the antibiotic ciprofloxacin, which is known to be effective against systemic salmonella infection. Maglianes et al., 1993, Antimicrobial Agen.
ts Chemo. 37: 2293.

The experimental group and control group of Balbc mice were orally inoculated with SL1344lux. Eight days after inoculation, 100 mg ciprofloxacin hydrochloride (3 m
g / kg-body weight; Sigma Chemical, St. Louis, MO)
Was injected intraperitoneally. After treatment of the experimental group, both groups of animals were imaged several times (as described above) for 5.5 hours after treatment.

Typical images are shown in FIGS. FIGS. 10B and 10D are composite images of animals representing the control group and the treatment group, respectively, immediately before the treatment of the experimental group is started. FIG. 10C
And 10E are composite images of the same animal 5.5 hours after the start of treatment. Control bioluminescence was increased by a factor of 7.5, whereas bioluminescence on the abdomen of ciprofloxacin-treated animals decreased to undetectable levels during this period. The total number of photons detected on the abdominal area was determined, normalized to the value at t = 0, and plotted in FIG. 10A with respect to time after treatment.

This data shows that the methods and compositions of the present invention can be used to assess the effects of drugs on the spread of in vivo infections.

Although the invention has been described with respect to particular methods and embodiments, it will be understood that various changes and modifications can be made without departing from the invention.

FIG. 1 is a map of the lux pCGLS1 plasmid used to transform Salmonella SL1344, BJ66 and LB5000 strains to produce SL1344lux, BJ66lux and LB5000lux strains. FIG. 2A shows the results of an assay measuring adhesion and invasion of macrophages and HEp-2 cells by Salmonella SL1344lux and BJ66lux strains. FIG. 2A shows luminescent bacterial cells localized in the wells of the assay dish. A pseudo-color image obtained by integrating photons over 1 minute was superimposed on the gray scale image of the assay dish to create the indicated “composite image”. FIG. 2B shows the results of an assay measuring the adhesion and invasion of macrophages and HEp-2 cells by Salmonella SL1344lux and BJ66lux strains. FIG. 2B shows the relative light intensity of wells that were not treated with gentamicin. FIG. 2C shows the results of an assay measuring the adhesion and invasion of macrophages and HEp-2 cells by Salmonella SL1344lux and BJ66lux strains. FIG. 2C shows the number of colony forming units (CFU) per ml isolated from the same well imaged in FIG. 2B. FIG. 2D shows the results of an assay measuring adhesion and invasion of macrophages and HEp-2 cells by Salmonella SL1344lux and BJ66lux strains. FIG. 2D shows the relative light intensity of wells treated with gentamicin. FIG. 2E shows the results of an assay measuring adhesion and invasion of macrophages and HEp-2 cells by Salmonella SL1344lux and BJ66lux strains. FIG. 2E shows the number of colony forming units (CFU) per ml isolated from the same well imaged in FIG. 2D. FIG. 3A is a composite image of four glass capillaries containing a dilution of LB5000lux bacterial suspension. Luminescence was measured by integration for 30 seconds. There are air pockets on both sides of the suspension in each capillary. FIG. 3B represents the bioluminescence distribution after inoculation of wild type SL1344lux into the abdominal cavity of mice. FIG. 4 is a schematic diagram of a vial used to examine the transmission of light generated by LB5000lux through animal tissue. FIG. 5A is a composite image of Balb / c mice that were orally inoculated with low pathogenic LB5000lux Salmonella and imaged at the times indicated in the figure. The luminescent signal was integrated for 5 minutes. FIG. 5B is a synthetic image of Balb / c mice that were orally inoculated with low pathogenic LB5000lux Salmonella and imaged at the times indicated in the figure. The luminescent signal was integrated for 5 minutes. FIG. 5C is a composite image of Balb / c mice that were orally inoculated with non-invasive BJ66lux Salmonella and imaged at the times indicated in the figure. The luminescent signal was integrated for 5 minutes. FIG. 5D is a composite image of Balb / c mice that were orally inoculated with non-invasive BJ66lux Salmonella and imaged at the times indicated in the figure. The luminescent signal was integrated for 5 minutes. FIG. 5E is a composite image of Balb / c mice that were orally inoculated with pathogenic SL1344lux Salmonella and imaged at the times indicated in the figure. The luminescent signal was integrated for 5 minutes. FIG. 5F is a composite image of Balb / c mice that were orally inoculated with pathogenic SL1344lux Salmonella and imaged at the times indicated in the figure. The luminescent signal was integrated for 5 minutes. FIG. 6 shows the distribution of Salmonella in mice 32 hours after intraperitoneal (ip) injection of pathogenic SL1344lux bacterial strain (left 2) or low pathogenic SL5000lux bacterial strain (right 2). It is a composite image. Figures 7A and 7B show the distribution of pathogenic Salmonella in mice resistant to systemic Salmonella infection (129X Balb / c, Ityr ^{/ s} ). 7A is the first day, and FIG. 7B is the eighth day. FIG. 8A shows the distribution of Salmonella mutant (BJ66lux) with reduced pathogenicity 7 days after oral inoculation. FIG. 8A shows external non-invasive imaging of luminescence. Organs with symbols are C-cecum, L-liver, I-small intestine, Sp-spleen. FIG. 8B shows the distribution of Salmonella mutant (BJ66lux) with reduced pathogenicity 7 days after oral inoculation. FIG. 8B is an image of the same animal as that shown in FIG. Organs with symbols are C-cecum, L-liver, I-small intestine, Sp-spleen. FIG. 8C shows the distribution of Salmonella mutant (BJ66lux) with reduced pathogenicity 7 days after oral inoculation. Organs with symbols are C-cecum, L-liver, I-small intestine, Sp-spleen. FIG. 8C shows an image after an abdominal wall incision made after injecting air into the intestinal lumen in front and behind the cecum. FIG. 9A shows the distribution of Salmonella SL1344lux in susceptible Balb / c mice after intraperitoneal inoculation of SL1344lux. FIG. 9A was taken before opening the abdominal cavity. FIG. 9B shows the distribution of Salmonella SL1344lux in susceptible Balb / c mice after intraperitoneal inoculation of SL1344lux. FIG. 9B was taken after opening the abdominal cavity. FIG. 9C shows the distribution of Salmonella SL1344lux in susceptible Balb / c mice after intraperitoneal inoculation of SL1344lux. FIG. 9C was taken after the cecum was pulled to the left. FIG. 10A represents the effect of ciprofloxacin treatment on bioluminescence from SL1344lux Salmonella in orally inoculated mice. FIG. 10A is a graph representing the relative bioluminescence measured from the abdominal area as a function of time after the start of treatment for treated and untreated animals. FIG. 10B represents the effect of ciprofloxacin treatment on bioluminescence from SL1344lux Salmonella in orally inoculated mice. FIG. 10B is a composite image of a mouse 8 days after oral inoculation with SL1344lux Salmonella and before treatment with ciprofloxacin. FIG. 10C represents the effect of ciprofloxacin treatment on bioluminescence from SL1344lux Salmonella in orally inoculated mice. FIG. 10C is a composite image 5.5 hours after the same mouse control (no treatment). FIG. 10D represents the effect of ciprofloxacin treatment on bioluminescence from SL1344lux Salmonella in orally inoculated mice. FIG. 10D is a composite image of a mouse 8 days after oral inoculation with SL1344lux Salmonella and before treatment with ciprofloxacin. FIG. 10E represents the effect of ciprofloxacin treatment on bioluminescence from SL1344lux Salmonella in orally inoculated mice. FIG. 10E is a composite image of 5.5 hours (FIG. 10E) after treatment of the same mouse.

Claims (1)

A method or composition as described herein.

Images