U.S. patent application number 11/818207 was filed with the patent office on 2008-09-18 for non-invasive localization of a light-emitting conjugate in a mammal.
Invention is credited to David A. Benaron, Christopher H. Contag, Pamela R. Contag.
Application Number | 20080226563 11/818207 |
Document ID | / |
Family ID | 46304661 |
Filed Date | 2008-09-18 |
United States Patent
Application |
20080226563 |
Kind Code |
A1 |
Contag; Pamela R. ; et
al. |
September 18, 2008 |
Non-invasive localization of a light-emitting conjugate in a
mammal
Abstract
Methods and compositions for detecting and localizing light
originating from a mammal are disclosed. Also disclosed are methods
for targeting light emission to selected regions, as well as for
tracking entities within the mammal. In addition, animal models for
disease states are disclosed, as are methods for localizing and
tracking the progression of disease or a pathogen within the
animal, and for screening putative therapeutic compounds effective
to inhibit the disease or pathogen.
Inventors: |
Contag; Pamela R.; (San
Jose, CA) ; Contag; Christopher H.; (San Jose,
CA) ; Benaron; David A.; (Redwood City, CA) |
Correspondence
Address: |
ROBINS & PASTERNAK
1731 EMBARCADERO ROAD, SUITE 230
PALO ALTO
CA
94303
US
|
Family ID: |
46304661 |
Appl. No.: |
11/818207 |
Filed: |
June 13, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11143422 |
Jun 2, 2005 |
7255851 |
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11818207 |
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10319542 |
Dec 16, 2002 |
6923951 |
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11143422 |
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09233698 |
Jan 19, 1999 |
6649143 |
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10319542 |
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08602396 |
Feb 16, 1996 |
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09233698 |
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08270631 |
Jul 1, 1994 |
5650135 |
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08602396 |
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Current U.S.
Class: |
424/9.6 ;
424/9.1 |
Current CPC
Class: |
C12N 2830/60 20130101;
Y10S 436/80 20130101; C07K 14/195 20130101; G01N 33/582 20130101;
A01K 2227/105 20130101; A61K 49/0097 20130101; A01K 2217/05
20130101; A61K 49/0008 20130101; A01K 2267/0337 20130101; C12Q 1/02
20130101; C12Q 1/66 20130101; A61K 49/0045 20130101; A01K 2267/0393
20130101; A01K 67/0275 20130101 |
Class at
Publication: |
424/9.6 ;
424/9.1 |
International
Class: |
A61K 49/00 20060101
A61K049/00 |
Claims
1. A non-invasive method for detecting an entity under study from
within a mammalian subject, comprising: administering to the
mammalian subject a conjugate of the entity and a light-generating
moiety; and measuring photon emission through an opaque tissue of
said mammalian subject from the light-generating moiety.
2. The method of claim 1, wherein said measuring is done using a
photodetector device.
3. The method of claim 2, wherein said measuring is carried out
with an intensified charge-coupled photodetector device.
4. The method of claim 2, wherein said measuring is carried out
with a cooled charge-coupled photodetector device.
5. The method of claim 1, wherein said measuring is carried out
using fiber optic cables.
6. The method of claim 5, wherein said fiber optic cables terminate
in a tightly-packed array.
7. The method of claim 5, wherein said fiber optic cables detect
light from a limited defined region of the subject.
8. The method of claim 1, wherein the light-emitting moiety is a
fluorescent molecule.
9. The method of claim 8, wherein the fluorescent molecule is green
fluorescent protein, lumazine, or yellow fluorescent protein.
10. The method of claim 8, wherein an input of light to excite the
fluorescent molecule is produced by a laser.
11. The method of claim 1, wherein the light-emitting moiety is a
bioluminescent molecule.
12. The method of claim 11, wherein the bioluminescent molecule is
a luciferase protein.
13. The method of claim 1, further comprising: repeating said
measuring at selected intervals, wherein said repeating is
effective to track localization of the entity in the subject over
time.
14. The method of claim 1, further comprising administering a
compound to said subject, and measuring photon emission from said
subject after administration of said compound.
15. The method of claim 14, further comprising: repeating at
selected intervals said measuring after administration of said
compound, wherein said repeating is effective to track an effect of
said compound on a level of photon emission in said subject over
time.
16. The method of claim 1, wherein said method further comprises,
prior to said measuring, placing the subject in a detection field
of the photodetector device.
17. The method of claim 1, wherein photons that make up said photon
emission are visible light photons.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of
application Ser. No. 11/143,422 which is a divisional application
of application Ser. No. 10/319,542 filed Dec. 16, 2002, now
allowed, which is a divisional of application Ser. No. 09/233,698
filed Jan. 19, 1999, now U.S. Pat. No. 6,649,143, which is a
continuation of application Ser. No. 08/602,396 filed Feb. 16,
1999, now abandoned, which is a continuation-in-part application of
application Ser. No. 08/270,631 filed Jul. 1, 2994, now U.S. Pat.
No. 5,650,135, from which priority is claimed under 35 U.S.C.
.sctn.120, and which applications are herein incorporated by
reference in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to noninvasive methods and
compositions for detecting, localizing and tracking light-emitting
entities and biological events in a mammalian subject.
BACKGROUND OF THE INVENTION
[0003] The ability to monitor the progression of infectious
diseases is limited by the current ex vivo methods of detecting and
quantifying infectious agents in tissues. The replication of an
infectious agent in a host often involves primary, secondary and
tertiary sites of replication. The sites of replication and the
course that an infectious agent follows through these sites is
determined by the route of inoculation, factors encoded by the host
as well as determinants of the infecting agent.
[0004] Experience may offer, in some cases, an estimate of probable
sites of replication and the progress of an infection. It is more
often the case, however, that the sites of infection, and the pace
of the disease are either not known or can only roughly be
estimated. Moreover, the progression of an infectious disease, even
in inbred strains of mice, is often individualized, and serial, ex
vivo analyses of many infected animals need to be conducted to
determine, on the average, what course a disease will follow in an
experimentally infected host.
[0005] Accordingly, it would be desirable to have a means of
tracking the progression of infection in an animal model. Ideally,
the tracking could be done non-invasively, such that a single
animal could be evaluated as often as necessary without detrimental
effects. Methods and compositions of the present invention provide
a non-invasive approach to detect, localize and track a pathogen,
as well as other entities, in a living host, such as a mammal.
III. SUMMARY OF THE INVENTION
[0006] In one embodiment, the invention includes a noninvasive
method for detecting the localization of a biocompatible entity in
a mammalian subject. The entity can be a molecule, macromolecule,
cell, microorganism (including a pathogen), a particle, or the
like.
[0007] The method includes administering to the subject a conjugate
of the entity and a light-generating moiety. Light-generating
moieties are typically molecules or macromolecules that give off
light. They may generate light as a result of radiation absorption
(e.g., fluorescent or phosphorescent molecules), or as a result of
a chemical reaction. (e.g., bioluminescent proteins). Exemplary
light-generating moieties are bioluminescent proteins, such as
luciferase and aequorin, and colored or fluorescent proteins, such
as yellow fluorescent protein and ferredoxin IV.
[0008] The moiety may be conjugated to the entity by a variety of
techniques, including incorporation during synthesis of the entity
(e.g., chemical or genetic, such a fusion protein of an antibody
fragment and a light-generating protein), chemical coupling
post-synthesis, non-covalent association (e.g., encapsulation by
liposomes), in-situ synthesis in the entity (e.g., expression of a
heterologous bioluminescent protein in a transformed cell), or in
situ activatable promoter-controlled expression of a bioluminescent
protein in cells of a transgenic animal stimulated by a promoter
inducer (e.g., interferon-activated promoter stimulated by
infection with a virus).
[0009] After a period of time in which the conjugate can localize
in the subject, the subject is immobilized within the detection
field of a photodetector device for a period of time effective to
measure a sufficient amount of photon emission (with the
photodetector device) to construct an image. An exemplary
photodetector device is an intensified charge-coupled device (ICCD)
camera coupled to an image processor. If the image can be
constructed in a time short relative to the time scale at which an
"unimmobilized" subject moves, the subject is inherently
"immobilized" during imaging and no special immobilization
precautions are required. An image from the photon emission data is
then constructed.
[0010] The method described above can be used to track the
localization of the entity in the subject over time, by repeating
the imaging steps at selected intervals and constructing images
corresponding to each of those intervals.
[0011] The method described above can be used in a number of
specific applications, by attaching, conjugating or incorporating
targeting moieties onto the entity. The targeting moiety may be an
inherent property of the entity (e.g., antibody or antibody
fragment), or it may be conjugated to, attached to, or incorporated
in the entity (e.g., liposomes containing antibodies). Examples of
targeting moieties include antibodies, antibody fragments, enzyme
inhibitors, receptor-binding molecules, various toxins and the
like. Targets of the targeting moiety may include sites of
inflammation, infection, thrombotic plaques and tumor cells.
Markers distinguishing these targets, suitable for recognition by
targeting moieties, are well known.
[0012] Further, the method may be used to detect and localize sites
of infection by a pathogen in an animal model, using the pathogen
(e.g., Salmonella) conjugated to a light-generating moiety as the
entity.
[0013] In a related embodiment, the invention includes a
noninvasive method for detecting the level of a biocompatible
entity in a mammalian subject over time. The method is similar to
methods described above, but is designed to detect changes in the
level of the entity in the subject over time, without necessarily
localizing the entity in the form of an image. This method is
particularly useful for monitoring the effects of a therapeutic
substance, such an antibiotic, on the levels of an entity, such as
a light-emitting bacterium, over time.
[0014] In another embodiment, the invention includes a noninvasive
method for detecting the integration of a transgene in a mammalian
subject. The method includes administering to the subject, a vector
construct effective to integrate a transgene into mammalian cells.
Such constructs are well known in the art. In addition to the
elements necessary to integrate effectively, the construct contains
a transgene (e.g., a therapeutic gene), and a gene encoding a
light-generating protein under the control of a selected
activatable promoter. After a period of time in which the construct
can achieve integration, the promoter is activated. For example, if
an interferon inducible promoter is used, a poly-inosine and
-cytosine duplex (poly-IC) can be locally administered (e.g.,
footpad injection) to stimulate interferon production. The HIV LTR
could similarly be used and induced, for example, with
dimethylsulfoxide (DMSO). The subject is then placed within the
detection field of a photodetector device, such as an individual
wearing light-intensifying "night vision" goggles, and the level of
photon emission is measured, or evaluated. If the level is above
background (i.e., if light can be preferentially detected in the
"activated" region), the subject is scored as having integrated the
transgene.
[0015] In a related embodiment, the invention includes a
noninvasive method for detecting the localization of a
promoter-induction event in an animal made transgenic or chimeric
for a construct including a gene encoding a light-generating
protein under the control of an inducible promoter. Promoter
induction events include the administration of a substance which
directly activates the promoter, the administration of a substance
which stimulates production of an endogenous promoter activator
(e.g., stimulation of interferon production by RNA virus
infection), the imposition of conditions resulting in the
production of an endogenous promoter activator (e.g., heat shock or
stress), and the like. The event is triggered, and the animal is
imaged as described above.
[0016] In yet another embodiment, the invention includes pathogens,
such as Salmonella, transformed with a gene expressing a
light-generating protein, such as luciferase.
[0017] In another aspect, the invention includes a method of
identifying therapeutic compounds effective to inhibit spread of
infection by a pathogen. The method includes administering a
conjugate of the pathogen and a light-generating moiety to control
and experimental animals, treating the experimental animals with a
putative therapeutic compound, localizing the light-emitting
pathogen in both control and experimental animals by the methods
described above, and identifying the compound as therapeutic if the
compound is effective to significantly inhibit the spread or
replication of the pathogen in the experimental animals relative to
control animals. The conjugates include a fluorescently-labeled
antibodies, fluorescently-labeled particles, fluorescently-labeled
small molecules, and the like.
[0018] In still another aspect, the invention includes a method of
localizing entities conjugated to light-generating moieties through
media of varying opacity. The method includes the use of
photodetector device to detect photons transmitted through the
medium, integrate the photons over time, and generate an image
based on the integrated signal.
[0019] In yet another embodiment, the invention includes a method
of measuring the concentration of selected substances, such as
dissolved oxygen or calcium, at specific sites in an organism. The
method includes entities, such as cells, containing a concentration
sensor--a light-generating molecule whose ability to generate light
is dependent on the concentration of the selected substance. The
entity containing the light-generating molecule is administered
such that it adopts a substantially uniform distribution in the
animal or in a specific tissue or organ system (e.g., spleen). The
organism is imaged, and the intensity and localization of light
emission is correlated to the concentration and location of the
selected substance. Alternatively, the entity contains a second
marker, such as a molecule capable of generating light at a
wavelength other than the concentration sensor. The second marker
is used to normalize for any non-uniformities in the distribution
of the entity in the host, and thus permit a more accurate
determination of the concentration of the selected substance.
[0020] In another aspect, the invention includes a method of
identifying therapeutic compounds effective to inhibit the growth
and/or the metastatic spread of a tumor. The method includes (i)
administering tumor cells labeled with or containing
light-generating moieties to groups of experimental and control
animals, (ii) treating the experimental group with a selected
compound, (iii) localizing the tumor cells in animals from both
groups by imaging photon emission from the light-generating
molecules associated with the tumor cells with a photodetector
device, and (iv) identifying a compound as therapeutic if the
compound is able to significantly inhibit the growth and/or
metastatic spread of the tumor in the experimental group relative
to the control group.
[0021] These and other objects and features of the invention will
be more fully appreciated when the following detailed description
of the invention is read in conjunction with the accompanying
drawings.
IV. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIGS. 1A, 1B and 1C show a map of the lux pCGLS1 plasmid
used to transform Salmonella strains SL1344, BJ66 and LB5000 to
generate strains SL1344lux, BJ66lux and LB5000lux. FIG. 1A depicts
a restriction enzyme map of the lux operon, which is inserted into
the BamHI site of the polylinker depicted in FIG. 1B. A sequence
included in the multiple cloning site (MCS) is provided in FIG. 1B,
with the Bam HI site indicated in bold type. A graphical
representation of a pUC18 vector without insert is shown in FIG.
1C.
[0023] FIGS. 2A, 2B, 2C, 2D and 2E depict the adherence and
invasion of Salmonella strains SL1344lux and BJ66lux on macrophages
and HEp-2 cells.
[0024] FIG. 2A depicts luminescent bacterial cells localized in
wells of an assay dish. The pseudo-color image, obtained by
integrating photons over one minute, is superimposed over a gray
scale image of the assay dish, producing the "composite image"
shown.
[0025] FIG. 2B depicts the relative light intensity of wells that
were not treated with gentamicin.
[0026] FIG. 2C depicts the number of colony forming units (CFU) per
ml isolated from the same wells as were imaged in FIG. 2B.
[0027] FIG. 2D depicts the relative light intensity of wells that
were treated with gentamicin.
[0028] FIG. 2E depicts the number of colony forming units (CFU) per
ml isolated from the same wells as were imaged in FIG. 2D.
[0029] FIG. 3A depicts a composite image of four glass capillary
tubes containing dilutions of LB5000lux bacterial suspensions.
Luminescence was determined by integrating over 30 seconds. Air
pockets are present in each tube on both sides of the
suspension.
[0030] FIG. 3B depicts the distribution of bioluminescence
following intraperitoneal inoculation of wild-type SL1344lux into
mice.
[0031] FIG. 4 depicts the effect of human blood on the light
emission from bioluminescent Salmonella.
[0032] FIG. 5 depicts a schematic diagram of a vial used to test
the transmission of light generated by LB5000lux through animal
tissue.
[0033] FIG. 6A depicts composite images of Balb/c mice orally
inoculated with low virulence LB5000lux Salmonella, and imaged at
the times indicated. The luminescence signal was integrated over 5
minutes.
[0034] FIG. 6B depicts composite images of Balb/c mice orally
inoculated with non-invasive BJ66lux Salmonella, and imaged at the
times indicated. The luminescence signal was integrated over 5
minutes.
[0035] FIG. 6C depicts composite images of Balb/c mice orally
inoculated with virulent SL1344lux Salmonella, and imaged at the
times indicated. The luminescence signal was integrated over 5
minutes.
[0036] FIG. 7 depicts a composite image showing the distribution of
Salmonella in mice 32 hours following intraperitoneal (i.p.)
injections with either virulent SL1344lux (left two animals) or low
virulence LB5000lux (right two animals) strains of the
bacterium.
[0037] FIG. 8A depicts the distribution of virulent Salmonella in
mice resistant to systemic Salmonella infections (129.times.Balb/c,
Ity.sup.r/s) on day one (1).
[0038] FIG. 8B depicts the distribution of virulent Salmonella in
mice resistant to systemic Salmonella infections (129.times.Balb/c,
Ity.sup.r/s) on day eight (8).
[0039] FIGS. 9A, 9B, and 9C depict the distribution of mutant
Salmonella with reduced virulence (BJ66lux) seven days following
oral inoculation.
[0040] FIG. 9A depicts external, non-invasive imaging of the
luminescence.
[0041] FIG. 9B depicts the same animal imaged following laparotomy.
Labeled organs are cecum (C), liver (L), small intestine (I), and
spleen (Sp).
[0042] FIG. 9C depicts a post-laparotomy image generated following
injection of air into the lumen of the intestine both anterior and
posterior to the cecum.
[0043] FIGS. 10A, 10B and 10C depict the distribution of Salmonella
SL1344lux in susceptible Balb/c mice following intraperitoneal
inoculation with SL1344lux.
[0044] FIG. 10A depicts an image prior to the opening of the
peritoneal cavity.
[0045] FIG. 10B depicts an image after the opening of the
peritoneal cavity.
[0046] FIG. 10C depicts an image after the cecum was pulled to the
left side.
[0047] FIGS. 11A, 11B, 11C, 11D, and 11E depict the effects of
ciprofloxacin treatment on bioluminescence from SL1344lux
Salmonella in orally-inoculated mice.
[0048] FIG. 11A shows a graph of the relative bioluminescence
intensity, measured from the abdominal area, as a function of time
after initiation of treatment, for treated and untreated
animals.
[0049] FIGS. 11B and 11D depict composite images of mice 8 days
after oral inoculation with SL1344lux Salmonella, before treatment
with ciprofloxacin.
[0050] FIGS. 11C and 11E depict composite images of the same mice
5.5 hours either following treatment (FIG. 11E) or control (no
treatment: FIG. 11C).
[0051] FIG. 12 depicts bioluminescence as a reporter for
replication of HIV-1 in culture. The gray scale image of the plates
at 24 h, 60 h, 96 h, and 7 d, as indicated, is shown.
[0052] FIG. 13 depicts an assessment of the promoter activity in
tissues of transgenic mice containing a construct composed of the
regulatory portion of the HIV LTR (U3 region) upstream of the
coding sequence of the firefly luciferase gene. NRE, negative
response element; ENH, enhancer region, TAR-transactivation
responsive element.
[0053] FIG. 14 depicts topical delivery of substrate to dermal
cells in transgenic mice containing a construct composed of the
regulatory portion of the HIV LTR upstream of the coding sequence
of the firefly luciferase gene.
[0054] FIG. 15 depicts bioluminescence from induced ears as result
of topical luciferin delivery in transgenic mice containing a
construct composed of the regulatory portion of the HIV LTR
upstream of the coding sequence of the firefly luciferase gene.
[0055] FIG. 16 depicts unilateral induction of luciferase
expression in transgenic mice; the left half of the shaved dorsal
surface of the mice and the left ear were treated with DMSO to
activate expression of the HIV-1 LTR; luciferin was applied
topically over the entire surface of the back and both ears.
[0056] FIG. 17 depicts the detection of bioluminescence from
internal tissues in transgenic mice.
[0057] FIG. 18 depicts imaging of the abdomen of animals following
laprotomy demonstrating signals to localize the origin of internal
bioluminescence.
[0058] FIG. 19 depicts expression of the differential expression of
HIV-LTR in neonatal transgenic mice.
V. DETAILED DESCRIPTION OF THE INVENTION
[0059] A. Definitions
[0060] Unless otherwise indicated, all terms used herein have the
same meaning as they would to one skilled in the art of the present
invention.
[0061] Opaque medium is used herein to refer to a medium that is
"traditionally" opaque, not necessarily absolutely opaque.
Accordingly, an opaque medium is defined as a medium that is
commonly considered to be neither transparent nor translucent, and
includes items such as a wood board, and flesh and skin of a
mammal.
[0062] Luciferase, unless stated otherwise, includes prokaryotic
and eukaryotic luciferases, as well as variants possessing varied
or altered optical properties, such as luciferases that luminesce
at wavelengths in the red range.
[0063] Biocompatible entity is an entity that can be administered
to a mammal. This includes pathogens which may be deleterious to
the mammal. In reference to an animal whose cells contain a
transgene expressing a light-generating protein, biocompatible
entity refers to the transgene-containing cells comprising the
mammal.
[0064] Light-generating is defined as capable of generating light
through a chemical reaction or through the absorption of
radiation.
[0065] Light is defined herein, unless stated otherwise, as
electromagnetic radiation having a wavelength of between about 300
nm and about 1100 nm.
[0066] Spread of infection typically refers to the spreading and
colonization by a pathogen of host sites other than the initial
infection site. The term can also include, however, growth in size
and/or number of the pathogen at the initial infection site.
[0067] lux--prokaryotic genes associated with luciferase and photon
emission.
[0068] luc--eukaryotic genes associated with luciferase and photon
emission.
[0069] Promoter induction event refers to an event that results in
the direct or indirect induction of a selected inducible
promoter.
[0070] Heterologous gene refers to a gene which has been
transfected into a host organism. Typically, a heterologous gene
refers to a gene that is not originally derived from the
transfected or transformed cells' genomic DNA.
[0071] Transgene refers to a heterologous gene which has been
introduced, transiently or permanently, into the germ line or
somatic cells of an organism.
[0072] B. General Overview of the Invention
[0073] The present invention includes methods and compositions
relating to non-invasive imaging and/or detecting of light-emitting
conjugates in mammalian subjects. The conjugates contain a
biocompatible entity and a light-generating moiety. Biocompatible
entities include, but are not limited to, small molecules such as
cyclic organic molecules; macromolecules such as proteins;
microorganisms such as viruses, bacteria, yeast and fungi;
eukaryotic cells; all types of pathogens and pathogenic substances;
and particles such as beads and liposomes. In another aspect,
biocompatible entities may be all or some of the cells that
constitute the mammalian subject being imaged.
[0074] Light-emitting capability is conferred on the entities by
the conjugation of a light-generating moiety. Such moieties include
fluorescent molecules, fluorescent proteins, enzymatic reactions
giving off photons and luminescent substances, such as
bioluminescent proteins. The conjugation may involve a chemical
coupling step, genetic engineering of a fusion protein, or the
transformation of a cell, microorganism or animal to express a
bioluminescent protein. For example, in the case where the entities
are the cells constituting the mammalian subject being imaged, the
light-generating moiety may be a bioluminescent or fluorescent
protein "conjugated" to the cells through localized,
promoter-controlled expression from a vector construct introduced
into the cells by having made a transgenic or chimeric animal.
[0075] Light-emitting conjugates are typically administered to a
subject by any of a variety of methods, allowed to localize within
the subject, and imaged. Since the imaging, or measuring photon
emission from the subject, may last up to tens of minutes, the
subject is usually, but not always, immobilized during the imaging
process.
[0076] Imaging of the light-emitting entities involves the use of a
photodetector capable of detecting extremely low levels of
light--typically single photon events--and integrating photon
emission until an image can be constructed. Examples of such
sensitive photodetectors include devices that intensify the single
photon events before the events are detected by a camera, and
cameras (cooled, for example, with liquid nitrogen) that are
capable of detecting single photons over the background noise
inherent in a detection system.
[0077] Once a photon emission image is generated, it is typically
superimposed on a "normal" reflected light image of the subject to
provide a frame of reference for the source of the emitted photons
(i.e., localize the light-emitting conjugates with respect to the
subject). Such a "composite" image is then analyzed to determine
the location and/or amount of a target in the subject.
[0078] The steps and embodiments outlined above are presented in
greater detail, below.
[0079] C. Light-Emitting Entities
[0080] 1. Light-Generating Moieties.
[0081] The light-generating moieties (LGMs), molecules or
constructs useful in the practice of the present invention may take
any of a variety of forms, depending on the application. They share
the characteristic that they are luminescent, that is, that they
emit electromagnetic radiation in ultraviolet (UV), visible and/or
infra-red (IR) from atoms or molecules as a result of the
transition of an electronically excited state to a lower energy
state, usually the ground state.
[0082] Examples of light-generating moieties include
photoluminescent molecules, such as fluorescent molecules,
chemiluminescent compounds, phosphorescent compounds, and
bioluminescent compounds.
[0083] Two characteristics of LGMs that bear considerable relevance
to the present invention are their size and their spectral
properties. Both are discussed in the context of specific types of
light-generating moieties described below, following a general
discussion of spectral properties.
[0084] Spectral Properties. An important aspect of the present
invention is the selection of light-generating moieties that
produce light capable of penetrating animal tissue such that it can
be detected externally in a non-invasive manner. The ability of
light to pass through a medium such as animal tissue (composed
mostly of water) is determined primarily by the light's intensity
and wavelength.
[0085] The more intense the light produced in a unit volume, the
easier the light will be to detect. The intensity of light produced
in a unit volume depends on the spectral characteristics of
individual LGMs, discussed below, and on the concentration of those
moieties in the unit volume. Accordingly, conjugation schemes that
place a high concentration of LGMs in or on an entity (such as
high-efficiency loading of a liposome or high-level expression of a
bioluminescent protein in a cell) typically produce brighter
light-emitting conjugates (LECs) which are easier to detect through
deeper layers of tissue, than schemes which conjugate, for example,
only a single LGM onto each entity.
[0086] A second factor governing the detectability of an LGM
through a layer of tissue is the wavelength of the emitted light.
Water may be used to approximate the absorption characteristics of
animal tissue, since most tissues are composed primarily of water.
It is well known that water transmits longer-wavelength light (in
the red range) more readily than it does shorter wavelength
light.
[0087] Accordingly, LGMs which emit light in the range of yellow to
red (550-1100 nm) are typically preferable to LGMs which emit at
shorter wavelengths. Several of the LGMs discussed below emit in
this range. However, it will be noted, based on experiments
performed in support of the present invention and presented below,
that excellent results can be achieved in practicing the present
invention with LGMs that emit in the range of 486 nm, despite the
fact that this is not an optimal emission wavelength. These results
are possible, in part, due to the relatively high concentration of
LGMs (luciferase molecules) present in the LECs (transformed
Salmonella cells) used in these experiments, and to the use of a
sensitive detector. It will be understood that through the use of
LGMs with a more optimal emission wavelength, similar detection
results can be obtained with LGEs having lower concentrations of
the LGMs.
[0088] Fluorescence-based Moieties. Fluorescence is the
luminescence of a substance from a single electronically excited
state, which is of very short duration after removal of the source
of radiation. The wavelength of the emitted fluorescence light is
longer than that of the exciting illumination (Stokes' Law),
because part of the exciting light is converted into heat by the
fluorescent molecule.
[0089] Because fluorescent molecules require input of light in
order to luminesce, their use in the present invention may be more
complicated than the use of bioluminescent molecules. Precautions
are typically taken to shield the excitatory light so as not to
contaminate the fluorescence photon signal being detected from the
subject. Obvious precautions include the placement of an excitation
filter, such that employed in fluorescence microscope, at the
radiation source. An appropriately-selected excitation filter
blocks the majority of photons having a wavelength similar to that
of the photons emitted by the fluorescent moiety. Similarly a
barrier filter is employed at the detector to screen out most of
the photons having wavelengths other than that of the fluorescence
photons. Filters such as those described above can be obtained from
a variety of commercial sources, including Omega Optical, Inc.
(Brattleboro, Vt.).
[0090] Alternatively, a laser producing high intensity light near
the appropriate excitation wavelength, but not near the
fluorescence emission wavelength, can be used to excite the
fluorescent moieties. An x-y translation mechanism may be employed
so that the laser can scan the subject, for example, as in a
confocal microscope.
[0091] As an additional precaution, the radiation source can be
placed behind the subject and shielded, such that the only
radiation photons reaching the site of the detector are those that
pass all the way through the subject. Furthermore, detectors may be
selected that have a reduced sensitivity to wavelengths of light
used to excite the fluorescent moiety.
[0092] Through judicious application of the precautions above, the
detection of fluorescent LGMs according to methods of the present
invention is possible.
[0093] Fluorescent moieties include small fluorescent molecules,
such as fluorescein, as well as fluorescent proteins, such as green
fluorescent protein (Chalfie, et al., 1994, Science 263:802-805.,
Morin and Hastings, 1971, J. Cell. Physiol. 77:313) and lumazine
and yellow fluorescent proteins (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), whose fluorescence
characteristics have not been evaluated, may be fluorescent and
thus applicable for use with the present invention. Ferredoxin IV
is a particularly promising candidate, as it has a reddish color,
indicating that it may fluoresce or reflect at a relatively long
wavelength and produce light that is effective at penetrating
tissue. Furthermore, the molecule is small for a protein (95 amino
acids), and can thus be conjugated to entities with a minimal
impact on their function.
[0094] An advantage of small fluorescent molecules is that they are
less likely to interfere with the bioactivity of the entity to
which they are attached than a would a larger light-generating
moiety. In addition, commercially-available fluorescent molecules
can be obtained with a variety of excitation and emission spectra
that are suitable for use with the present invention. For example,
Molecular Probes (Eugene, Oreg.) sells a number of fluorophores,
including Lucifer Yellow (abs. at 428 nm, and emits at 535 nm) and
Nile Red (abs. at 551 nm and emits at 636 nm). Further, the
molecules can be obtained derivatized with a variety of groups for
use with various conjugation schemes (e.g., from Molecular
Probes).
[0095] Bioluminescence-Based Moieties. The subjects of
chemiluminescence (luminescence as a result of a chemical reaction)
and bioluminescence (visible luminescence from living organisms)
have, in many aspects, been thoroughly studied (e.g., Campbell,
1988, Chemiluminescence, Principles and Applications in Biology and
Medicine (Chichester, England: Ellis Horwood Ltd. and VCH
Verlagsgesellschaft mbH). A brief summary of salient features
follows.
[0096] Bioluminescent molecules are distinguished from fluorescent
molecules in that they do not require the input of radiative energy
to emit light. Rather, bioluminescent molecules utilize chemical
energy, such as ATP, to produce light. An advantage of
bioluminescent moieties, as opposed to fluorescent moieties, is
that there is virtually no background in the signal. The only light
detected is light that is produced by the exogenous bioluminescent
moiety. In contrast, the light used to excite a fluorescent
molecule often results in the fluorescence of substances other than
the intended target. This is particularly true when the
"background" is as complex as the internal environment of a living
animal.
[0097] Several types of bioluminescent molecules are known. They
include the luciferase family (e.g., Wood, et al., 1989, Science
244:700-702) and the aequorin family (e.g., Prasher, et al.,
Biochem. 26:1326-1332). Members of the luciferase family have been
identified in a variety of prokaryotic and eukaryotic organisms.
Luciferase and other enzymes involved in the prokaryotic
luminescent (lux) systems, as well as the corresponding lux genes,
have been isolated from marine bacteria in the Vibrio and
Photobacterium genera and from terrestrial bacteria in the
Xenorhabdus genus.
[0098] An exemplary eukaryotic organism containing a luciferase
system (luc) is the North American firefly Photinus pyralis.
Firefly luciferase has been extensively studied, and is widely used
in ATP assays. cDNAs encoding luciferases from Pyrophorus
plagiophthalamus, another species of click beetle, have been cloned
and expressed (Wood, et al., 1989, Science 244:700-702). This
beetle is unusual in that different members of the species emit
bioluminescence of different colors. Four classes of clones, having
95-99% homology with each other, were isolated. They emit light at
546 nm (green), 560 nm (yellow-green), 578 nm (yellow) and 593 nm
(orange). The last class (593 nm) may be particularly advantageous
for use as a light-generating moiety with the present invention,
because the emitted light has a wavelength that penetrates tissues
more easily than shorter wavelength light.
[0099] Luciferases, as well as aequorin-like molecules, require a
source of energy, such as ATP, NAD(P)H, and the like, and a
substrate, such as luciferin or coelentrizine and oxygen.
[0100] The substrate luciferin must be supplied to the luciferase
enzyme in order for it to luminesce. In those cases where a
luciferase enzyme is introduced as an expression product of a
vector containing cDNA encoding a lux luciferase, a convenient
method for providing luciferin is to express not only the
luciferase but also the biosynthetic enzymes for the synthesis of
luciferin. In cells transformed with such a construct, oxygen is
the only extrinsic requirement for bioluminescence. Such an
approach, detailed in Example 1, is employed to generate
lux-transformed Salmonella, which are used in experiments performed
in support of the present invention and detailed herein.
[0101] The plasmid construct, encoding the lux operon obtained from
the soil bacterium Xenorhabdus luminescens (Frackman, et al., 1990,
J. Bact. 172:5767-5773), confers on transformed E. coli the ability
to emit photons through the expression of the two subunits of the
heterodimeric luciferase and three accessory proteins (Frackman, et
al., 1990). Optimal bioluminescence for E. Coli expressing the lux
genes of X. luminescens is observed at 37.degree. C. (Szittner and
Meighen, 1990, J. Biol. Chem. 265:16581-16587, Xi, et al., 1991, J.
Bact. 173:1399-1405) in contrast to the low temperature optima of
luciferases from eukaryotic and other prokaryotic luminescent
organisms (Campbell, 1988, Chemiluminescence. Principles and
Applications in Biology and Medicine (Chichester, England: Ellis
Horwood Ltd. and VCH Verlagsgesellschaft mbH)). The luciferase from
X. luminescens, therefore, is well-suited for use as a marker for
studies in animals.
[0102] Luciferase vector constructs such as the one described above
and in Example 1, can be adapted for use in transforming a variety
of host cells, including most bacteria, and many eukaryotic cells
(luc constructs). In addition, certain viruses, such as herpes
virus and vaccinia virus, can be genetically-engineered to express
luciferase. For example, Kovacs Sz. and Mettenlieter, 1991, J. Gen.
Virol. 72:2999-3008, teach the stable expression of the gene
encoding firefly luciferase in a herpes virus. Brasier and Ron,
1992, Meth. in Enzymol. 216:386-396, teach the use of luciferase
gene constructs in mammalian cells. Luciferase expression from
mammalian cells in culture has been studied using CCD imaging both
macroscopically (Israel and Honigman, 1991, Gene 104:139-145) and
microscopically (Hooper, et al., 1990, J. Biolum. and Chemilum.
5:123-130).
[0103] 2. Entities
[0104] The invention includes entities which have been modified or
conjugated to include a light-generating moiety, construct or
molecule, such as described above. Such conjugated or modified
entities are referred to as light-emitting entities, light-emitting
conjugates (LECS) or simply conjugates. The entities themselves may
take the form of, for example, molecules, macromolecules,
particles, microorganisms, or cells. The methods used to conjugate
a light-generating moiety to an entity depend on the nature of the
moiety and the entity. Exemplary conjugation methods are discussed
in the context of the entities described below.
[0105] Small molecules. Small molecule entities which may be useful
in the practice of the present invention include compounds which
specifically interact with a pathogen or an endogenous ligand or
receptor. Examples of such molecules include, but are not limited
to, drugs or therapeutic compounds; toxins, such as those present
in the venoms of poisonous organisms, including certain species of
spiders, snakes, scorpions, dinoflagellates, marine snails and
bacteria; growth factors, such as NGF, PDGF, TGF and TNF;
cytokines; and bioactive peptides.
[0106] The small molecules are preferably conjugated to
light-generating moieties that interfere only minimally, if at all,
with the bioactivity of the small molecule, such as small
fluorescent molecules (described above). Conjugations are typically
chemical in nature, and can be performed by any of a variety of
methods known to those skilled in the art.
[0107] The small molecule entity may be synthesized to contain a
light-generating moiety, so that no formal conjugation procedure is
necessary. Alternatively, the small molecule entity may be
synthesized with a reactive group that can react with the light
generating moiety, or vice versa.
[0108] Small molecules conjugated to light-generating moieties of
the present invention may be used either in animal models of human
conditions or diseases, or directly in human subjects to be
treated. For example, a small molecule which binds with high
affinity to receptor expressed on tumor cells may be used in an
animal model to localize and obtain size estimates of tumors, and
to monitor changes in tumor growth or metastasis following
treatment with a putative therapeutic agent. Such molecules may
also be used to monitor tumor characteristics, as described above,
in cancer patients.
[0109] Macromolecules. Macromolecules, such as polymers and
biopolymers, constitute another example of entities useful in
practicing the present invention. Exemplary macromolecules include
antibodies, antibody fragments, fusion proteins and certain vector
constructs.
[0110] Antibodies or antibody fragments, purchased from commercial
sources or made by methods known in the art (Harlow, et al., 1988,
Antibodies: A Laboratory Manual, Chapter 10, pg. 402, Cold Spring
Harbor Press), can be used to localize their antigen in a mammalian
subject by conjugating the antibodies to a light-generating moiety,
administering the conjugate to a subject by, for example,
injection, allowing the conjugate to localize to the site of the
antigen, and imaging the conjugate.
[0111] Antibodies and antibody fragments have several advantages
for use as entities in the present invention. By their nature, they
constitute their own targeting moieties. Further, their size makes
them amenable to conjugation with several types of light-generating
moieties, including small fluorescent molecules and fluorescent and
bioluminescent proteins, yet allows them to diffuse rapidly
relative to, for example, cells or liposomes.
[0112] The light-generating moieties can be conjugated directly to
the antibodies or fragments, or indirectly by using, for example, a
fluorescent secondary antibody. Direct conjugation can be
accomplished by standard chemical coupling of, for example, a
fluorophore to the antibody or antibody fragment, or through
genetic engineering. Chimeras, or fusion proteins can be
constructed which contain an antibody or antibody fragment coupled
to a fluorescent or bioluminescent protein. For example, Casadei,
et al., 1990, PNAS 87:2047-2051, describe a method of making a
vector construct capable of expressing a fusion protein of aequorin
and an antibody gene in mammalian cells.
[0113] Conjugates containing antibodies can be used in a number of
applications of the present invention. For example, a labeled
antibody directed against E-selectin, which is expressed at sites
of inflammation, can be used to localize the inflammation and to
monitor the effects of putative anti-inflammatory agents.
[0114] Vector constructs by themselves can also constitute
macromolecular entities applicable to the present invention. For
example, a eukaryotic expression vector can be constructed which
contains a therapeutic gene and a gene encoding a light-generating
molecule under the control of a selected promoter (i.e., a promoter
which is expressed in the cells targeted by the therapeutic gene).
Expression of the light-generating molecule, assayed using methods
of the present invention, can be used to determine the location and
level of expression of the therapeutic gene. This approach may be
particularly useful in cases where the expression of the
therapeutic gene has no immediate phenotype in the treated
individual or animal model.
[0115] Viruses. Another entity useful for certain aspects of the
invention are viruses. As many viruses are pathogens which infect
mammalian hosts, the viruses may be conjugated to a
light-generating moiety and used to study the initial site and
spread of infection. In addition, viruses labeled with a
light-generating moiety may be used to screen for drugs which
inhibit the infection or the spread of infection.
[0116] A virus may be labeled indirectly, either with an antibody
conjugated to a light-generating moiety, or by, for example,
biotinylating virions (e.g., by the method of Dhawan, et al., 1991,
J. Immunol. 147(1):102) and then exposing them to streptavidin
linked to a detectable moiety, such as a fluorescent molecule.
[0117] Alternatively, virions may be labeled directly with a
fluorophore like rhodamine, using, for example, the methods of Fan,
et al., 1992, J. Clin. Micro. 30(4):905. The virus can also be
genetically engineered to express a light-generating protein. The
genomes of certain viruses, such as herpes and vaccinia, are large
enough to accommodate genes as large as the lux or luc genes used
in experiments performed in support of the present invention.
[0118] Labeled virus can be used in animal models to localize and
monitor the progression of infection, as well as to screen for
drugs effective to inhibit the spread of infection. For example,
while herpes virus infections are manifested as skin lesions, this
virus can also cause herpes encephalitis. Such an infection can be
localized and monitored using a virus labeled by any of the methods
described above, and various antiviral agents can be tested for
efficacy in central nervous system (CNS) infections.
[0119] Particles. Particles, including beads, liposomes and the
like, constitute another entity useful in the practice of the
present invention. Due to their larger size, particles may be
conjugated with a larger number of light-generating molecules than,
for example, can small molecules. This results in a higher
concentration of light emission, which can be detected using
shorter exposures or through thicker layers of tissue. In addition,
liposomes can be constructed to contain an essentially pure
targeting moiety, or ligand, such as an antigen or an antibody, on
their surface. Further, the liposomes may be loaded with, for
example, bioluminescent protein molecules, to relatively high
concentrations (Campbell, 1988, Chemiluminescence. Principles and
Applications in Biology and Medicine (Chichester, England: Ellis
Horwood Ltd. and VCH Verlagsgesellschaft mbH)).
[0120] Furthermore, two types of liposomes may be targeted to the
same cell type such that light is generated only when both are
present. For example, one liposome may carry luciferase, while the
other carries luciferin. The liposomes may carry targeting
moieties, and the targeting moieties 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 a single or multiple cell surface
markers.
[0121] The liposomes are preferably surface-coated, e.g., by
incorporation of phospholipid-polyethyleneglycol conjugates, to
extend blood circulation time and allow for greater targeting via
the bloodstream. Liposomes of this type are well known.
[0122] Cells. Cells, both prokaryotic and eukaryotic, constitute
another entity useful in the practice of the present invention.
Like particles, cells can be loaded with relatively high
concentrations of light-generating moieties, but have the advantage
that the light-generating moieties can be provided by, for example,
a heterologous genetic construct used to transfect the cells. In
addition, cells can be selected that express "targeting moieties",
or molecules effective to target them to desired locations within
the subject. Alternatively, the cells can be transfected with a
vector construct expressing an appropriate targeting moiety.
[0123] The cell type used depends on the application. For example,
as is detailed below, bacterial cells, such as Salmonella, can be
used to study the infective process, and to evaluate the effects of
drugs or therapeutic agents on the infective process with a high
level of temporal and spatial resolution.
[0124] Bacterial cells constitute effective entities. For example,
they can be easily transfected to express a high levels of a
light-generating moiety, as well as high levels of a targeting
protein. In addition, it is possible to obtain E. coli libraries
containing bacteria expressing surface-bound antibodies which can
be screened to identify a colony expressing an antibody against a
selected antigen (Stratagene, La Jolla, Calif.). Bacteria from this
colony can then be transformed with a second plasmid containing a
gene for a light-generating protein, and transformants can be
utilized in the methods of the present invention, as described
above, to localize the antigen in a mammalian host.
[0125] Pathogenic bacteria can be conjugated to a light-generating
moiety and used in an animal model to follow the infection process
in vivo and to evaluate potential anti-infective drugs, such as new
antibiotics, for their efficacy in inhibiting the infection. An
example of this application is illustrated by experiments performed
in support of the present invention and detailed below.
[0126] Eukaryotic cells are also useful as entities in aspects of
the present invention. Appropriate expression vectors, containing
desired regulatory elements, are commercially available. The
vectors can be used to generate constructs capable of expressing
desired light-generating proteins in a variety of eukaryotic cells,
including primary culture cells, somatic cells, lymphatic cells,
etc. The cells can be used in transient expression studies, or, in
the case of cell lines, can be selected for stable
transformants.
[0127] Expression of the light-generating protein in transformed
cells can be regulated using any of a variety of selected
promoters. For example, if the cells are to be used as
light-emitting entities targeted to a site in the subject by an
expressed ligand or receptor, a constitutively-active promoter,
such as the CMV or SV40 promoter may be used. Cells transformed
with such a construct can also be used to assay for compounds that
inhibit light generation, for example, by killing the cells.
[0128] Alternatively, the transformed cells may be administered
such they become uniformly distributed in the subject, and express
the light-generating protein only under certain conditions, such as
upon infection by a virus or stimulation by a cytokine. Promoters
that respond to factors associated with these and other stimuli are
known in the art. In a related aspect, inducible promoters, such as
the Tet system (Gossen and Bujard, 1992, PNAS 89:5547-5551) can be
used to transiently activate expression of the light-generating
protein.
[0129] For example, CD4+ lymphatic cells can be transformed with a
construct containing tat-responsive HIV LTR elements, and used as
an assay for infection by HIV (Israel and Honigman, 1991, Gene
104:139-145). Cells transformed with such a construct can be
introduced into SCID-hu mice (McCune, et al., 1988, Science
241:1632-1639) and used as model for human HIV infection and
AIDS.
[0130] Tumor cell lines transformed as above, for example, with a
constitutively-active promoter, may be used to monitor the growth
and metastasis of tumors. Transformed tumor cells may be injected
into an animal model, allowed to form a tumor mass, and the size
and metastasis of the tumor mass monitored during treatment with
putative growth or metastasis inhibitors.
[0131] Tumor cells may also be generated from cells transformed
with constructs containing regulatable promoters, whose activity is
sensitive to various infective agents, or to therapeutic
compounds.
[0132] Cell Transformation. Transformation methods for both
prokaryotic cells and eukaryotic cells are well known in the art
(Sambrook, et al., 1989, In Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Vol. 2). Vectors containing
the appropriate regulatory elements and multiple cloning sites are
widely commercially available (e.g., Stratagene, La Jolla, Calif.,
Clontech, Palo Alto, Calif.).
[0133] D. Transgenic Animals Containing Genes Encoding
Light-Generating Proteins
[0134] In another aspect, the present invention includes transgenic
animals containing a heterologous gene construct encoding a
light-generating protein or complex of proteins.
[0135] The construct is driven by a selected promoter, and can
include, for example, various accessory proteins required for the
functional expression of the light-generating protein, as well as
selection markers and enhancer elements.
[0136] Activation of the promoter results in increased expression
of the genes encoding the light-generating molecules and accessory
proteins. Activation of the promoter is achieved by the interaction
of a selected biocompatible entity, or parts of the entity, with
the promoter elements. If the activation occurs only in a part of
the animal, only cells in that part will express the
light-generating protein.
[0137] For example, an interferon-inducible promoter, such as the
promoter for 3'-5' poly-A synthetase or the Mx protein (an
interferon-inducible promoter), can be used to detect the infection
of transgenic cells by a number of different RNA viruses.
[0138] In a related aspect, a promoter expressed in certain disease
states can be used to mark affected areas in a transgenic animal,
and expression of the light-generating moiety can be used to
monitor the effects of treatments for the disease state. For
example, E-selectin is expressed at sites of inflammation in vivo
(Pober and Cotran, 1991, Lab. Invest. 64:301-305). Accordingly, the
E-selectin promoter can be isolated and used to drive the
expression of a luciferase gene.
[0139] It is also possible to use methods of the invention with
tissue-specific promoters. This enables, for example, the screening
of compounds which are effective to inhibit pathogenic processes
resulting in the degeneration of a particular organ or tissue in
the body, and permits the tracking of cells (e.g., neurons) in, for
example, a developing animal.
[0140] Many promoters which are applicable for use with the present
invention are known in the art. In addition, methods are known for
isolating promoters of cloned genes, using information from the
gene's cDNA to isolate promoter-containing genomic DNA.
[0141] In a specific embodiment of the present invention,
transgenic animals expressing luciferase under the control of the
HIV-1 LTR have been generated. As demonstrated in specific
examples, luciferase expression serves as a real-time
bioluminescent reporter which allows the noninvasive assessment of
the level of promoter activity in vivo. As described, supra, the
photons from the in vivo luciferase reaction can be detected by a
CCD camera, after transmission through animal tissues, and used as
an indication of the level and location of gene expression both in
superficial and internal tissues.
[0142] E. Imaging of Light-Emitting Conjugates
[0143] Light emitting conjugates that have localized to their
intended sites in a subject may be imaged in a number of ways.
Guidelines for such imaging, as well as specific examples, are
described below.
[0144] 1. Localization of Light-Emitting Conjugates
[0145] In the case of "targeted" entities, that is, entities which
contain a targeting moiety--a molecule or feature designed to
localize the entity within a subject or animal at a particular site
or sites, localization refers to a state when an equilibrium
between bound, "localized", and unbound, "free" entities within a
subject has been essentially achieved. The rate at which such an
equilibrium is achieved depends upon the route of administration.
For example, a conjugate administered by intravenous injection to
localize thrombi may achieve localization, or accumulation at the
thrombi, within minutes of injection. On the other hand, a
conjugate administered orally to localize an infection in the
intestine may take hours to achieve localization.
[0146] Alternatively, localization may simply refer to the location
of the entity within the subject or animal at selected time periods
after the entity is administered. For example, in experiments
detailed herein, Salmonella are administered (e.g., orally) and
their spread is followed as a function of time. In this case, the
entity can be "localized" immediately following the oral
introduction, inasmuch as it marks the initial location of the
administered bacteria, and its subsequent spread or recession (also
"localization") may be followed by imaging.
[0147] In a related aspect, localization of, for example, injected
tumors cells expressing a light-generating moiety, may consist of
the cells colonizing a site within the animal and forming a tumor
mass.
[0148] By way of another example, localization is achieved when an
entity becomes distributed following administration. For example,
in the case of a conjugate administered to measure the oxygen
concentration in various organs throughout the subject or animal,
the conjugate becomes "localized", or informative, when it has
achieved an essentially steady-state of distribution in the subject
or animal.
[0149] In all of the above cases, a reasonable estimate of the time
to achieve localization may be made by one skilled in the art.
Furthermore, the state of localization as a function of time may be
followed by imaging the light-emitting conjugate according to the
methods of the invention.
[0150] 2. Photodetector Devices
[0151] An important aspect of the present invention is the
selection of a photodetector device with a high enough sensitivity
to enable the imaging of faint light from within a mammal in a
reasonable amount of time, preferably less than about 30 minutes,
and to use the signal from such a device to construct an image.
[0152] In cases where it is possible to use light-generating
moieties which are extremely bright, and/or to detect
light-emitting conjugates localized near the surface of the subject
or animal being imaged, a pair of "night-vision" goggles or a
standard high-sensitivity video camera, such as a Silicon
Intensified Tube (SIT) camera (e.g., Hamamatsu Photonic Systems,
Bridgewater, N.J.), may be used. More typically, however, a more
sensitive method of light detection is required.
[0153] In extremely low light levels, such as those encountered in
the practice of the present invention, the photon flux per unit
area becomes so low that the scene being imaged no longer appears
continuous. Instead, it is represented by individual photons which
are both temporally and spatially distinct form one another. Viewed
on a monitor, such an image appears as scintillating points of
light, each representing a single detected photon.
[0154] By accumulating these detected photons in a digital image
processor over time, an image can be acquired and constructed. In
contrast to conventional cameras where the signal at each image
point is assigned an intensity value, in photon counting imaging
the amplitude of the signal carries no significance. The objective
is to simply detect the presence of a signal (photon) and to count
the occurrence of the signal with respect to its position over
time.
[0155] At least two types of photodetector devices, described
below, can detect individual photons and generate a signal which
can be analyzed by an image processor.
[0156] Reduced-Noise Photodetection Devices. The first class
constitutes devices which achieve sensitivity by reducing the
background noise in the photon detector, as opposed to amplifying
the photon signal. Noise is reduced primarily by cooling the
detector array. The devices include charge coupled device (CCD)
cameras referred to as "backthinned", cooled CCD cameras. In the
more sensitive instruments, the cooling is achieved using, for
example, liquid nitrogen, which brings the temperature of the CCD
array to approximately -120.degree. C. The "backthinned" refers to
an ultra-thin backplate that reduces the path length that a photon
follows to be detected, thereby increasing the quantum efficiency.
A particularly sensitive backthinned cryogenic CCD camera is the
"TECH 512", a series 200 camera available from Photometrics, Ltd.
(Tucson, Ariz.).
[0157] Photon Amplification Devices. A second class of sensitive
photodetectors includes devices which amplify photons before they
hit the detection screen. This class includes CCD cameras with
intensifiers, such as microchannel intensifiers. A microchannel
intensifier typically contains a metal array of channels
perpendicular to and co-extensive with the detection screen of the
camera. The microchannel array is placed between the sample,
subject, or animal to be imaged, and the camera. Most of the
photons entering the channels of the array contact a side of a
channel before exiting. A voltage applied across the array results
in the release of many electrons from each photon collision. The
electrons from such a collision exit their channel of origin in a
"shotgun" pattern, and are detected by the camera.
[0158] Even greater sensitivity can be achieved by placing
intensifying microchannel arrays in series, so that electrons
generated in the first stage in turn result in an amplified signal
of electrons at the second stage. Increases in sensitivity,
however, are achieved at the expense of spatial resolution, which
decreases with each additional stage of amplification.
[0159] An exemplary microchannel intensifier-based single-photon
detection device is the C2400 series, available from Hamamatsu.
[0160] Image Processors. Signals generated by photodetector devices
which count photons need to be processed by an image processor in
order to construct an image which can be, for example, displayed on
a monitor or printed on a video printer. Such image processors are
typically sold as part of systems which include the sensitive
photon-counting cameras described above, and accordingly, are
available from the same sources (e.g., Photometrics, Ltd., and
Hamamatsu). Image processors from other vendors can also be used,
but more effort is generally required to achieve a functional
system.
[0161] The image processors are usually connected to a personal
computer, such as an IBM-compatible PC or an Apple Macintosh (Apple
Computer, Cupertino, Calif.), which may or may not be included as
part of a purchased imaging system. Once the images are in the form
of digital files, they can be manipulated by a variety of image
processing programs (such as "ADOBE PHOTOSHOP", Adobe Systems,
Adobe Systems, Mt. View, Calif.) and printed.
[0162] 3. Immobilizing Subject in Detection Field of Device
[0163] Detection Field Of Device. The detection field of the device
is defined as the area from which consistent measurements of photon
emission can be obtained.
[0164] In the case of a camera using an optical lens, the detection
field is simply the field of view accorded to the camera by the
lens. Similarly, if the photodetector device is a pair of "night
vision" goggles, the detection field is the field of view of the
goggles.
[0165] Alternatively, the detection field may be a surface defined
by the ends of fiber-optic cables arranged in a tightly-packed
array. The array is constructed to maximize the area covered by the
ends of the cables, as opposed to void space between cables, and
placed in close proximity to the subject. For instance, a clear
material such as plexiglass can be placed adjacent the subject, and
the array fastened adjacent the clear material, opposite from the
subject.
[0166] The fiber-optic cable ends opposite the array can be
connected directly to the detection or intensifying device, such as
the input end of a microchannel intensifier, eliminating the need
for a lens.
[0167] An advantage of this method is that scattering and/or loss
of photons is reduced by eliminating a large part of the air space
between the subject and the detector, and/or by eliminating the
lens. Even a high-transmission lens, such as the 60 mm AF Nikkor
macro lens used in experiments performed in support of the present
invention, transmits only a fraction of the light reaching the
front lens element.
[0168] With higher-intensity LGMs, photodiode arrays may be used to
measure photon emission. A photodiode array can be incorporated
into a relatively flexible sheet, enabling the practitioner to
partially "wrap" the array around the subject. This approach also
minimizes photon loss, and in addition, provides a means of
obtaining three-dimensional images of the bioluminescence.
[0169] Other approaches may be used to generate three-dimensional
images, including multiple detectors placed around the subject or a
scanning detector or detectors.
[0170] It will be understood that the entire animal or subject need
not necessarily be in the detection field of the photodetection
device. For example, if one is measuring a light-emitting conjugate
known to be localized in a particular region of the subject, only
light from that region, and a sufficient surrounding "dark" zone,
need be measured to obtain the desired information.
[0171] Immobilizing The Subject. In those cases where it is desired
to generate a two-dimensional or three-dimensional image of the
subject, the subject may be immobilized in the detection field of
the photodetection devices during the period that photon emission
is being measured. If the signal is sufficiently bright that an
image can be constructed from photon emission measured in less than
about 20 milliseconds, and the subject is not particularly
agitated, no special immobilization precautions may be required,
except to insure that the subject is in the field of the detection
device at the start of the measuring period.
[0172] If, on the other hand, the photon emission measurement takes
longer than about 20 msec, and the subject is agitated, precautions
to insure immobilization of the subject during photon emission
measurement, commensurate with the degree of agitation of the
subject, need to be considered to preserve the spatial information
in the constructed image. For example, in a case where the subject
is a person and photon emission measurement time is on the order of
a few seconds, the subject may simply be asked to remain as still
as possible during photon emission measurement (imaging). On the
other hand, if the subject is an animal, such as a mouse, the
subject can be immobilized using, for example, an anesthetic or a
mechanical restraining device.
[0173] A variety of restraining devices may be constructed. For
example, a restraining device effective to immobilize a mouse for
tens of seconds to minutes may be built by fastening a plexiglass
sheet over a foam cushion. The cushion has an indentation for the
animal's head at one end. The animal is placed under the plexiglass
such that its head is over the indentation, allowing it to breathe
freely, yet the movement of its body is constrained by the foam
cushion.
[0174] In cases where it is desired to measure only the total
amount of light emanating from a subject or animal, the subject
does not necessarily need to be immobilized, even for long periods
of photon emission measurements. All that is required is that the
subject be confined to the detection field of the photodetector
during imaging. It will be appreciated, however, that immobilizing
the subject during such measuring may improve the consistency of
results obtained, because the thickness of tissue through which
detected photons pass will be more uniform from animal to
animal.
[0175] 4. Further Considerations During Imaging
[0176] Fluorescent Light-Generating Moieties. The visualization of
fluorescent light-generating moieties requires an excitation light
source, as well as a photodetector. Furthermore, it will be
understood that the excitation light source is turned on during the
measuring of photon emission from the light-generating moiety.
[0177] Appropriate selection of a fluorophore, placement of the
light source and selection and placement of filters, all of which
facilitate the construction of an informative image, are discussed
above, in the section on fluorescent light-generating moieties.
[0178] High-Resolution Imaging. Photon scattering by tissue limits
the resolution that can be obtained by imaging LGMs through a
measurement of total photon emission. It will be understood that
the present invention also includes embodiments in which the
light-generation of LGMs is synchronized to an external source
which can be focused at selected points within the subject, but
which does not scatter significantly in tissue, allowing the
construction of higher-resolution images. For example, a focused
ultrasound signal can be used to scan, in three dimensions, the
subject being imaged. Light-generation from areas which are in the
focal point of the ultrasound can be resolved from other photon
emission by a characteristic oscillation imparted to the light by
the ultrasound (e.g., Houston and Moerner, U.S. Pat. No. 4,614,116,
issued 30 Sep. 1986.)
[0179] 5. Constructing an Image of Photon Emission
[0180] In cases where, due to an exceptionally bright
light-generating moiety and/or localization of light-emitting
conjugates near the surface of the subject, a pair of
"night-vision" goggles or a high sensitivity video camera was used
to obtain an image, the image is simply viewed or displayed on a
video monitor. If desired, the signal from a video camera can be
diverted through an image processor, which can store individual
video frames in memory for analysis or printing, and/or can
digitize the images for analysis and printing on a computer.
[0181] Alternatively, if a photon counting approach is used, the
measurement of photon emission generates an array of numbers,
representing the number of photons detected at each pixel location,
in the image processor. These numbers are used to generate an
image, typically by normalizing the photon counts (either to a
fixed, pre-selected value, or to the maximum number detected in any
pixel) and converting the normalized number to a brightness
(greyscale) or to a color (pseudocolor) that is displayed on a
monitor. In a pseudocolor representation, typical color assignments
are as follows. Pixels with zero photon counts are assigned black,
low counts blue, and increasing counts colors of increasing
wavelength, on up to red for the highest photon count values. The
location of colors on the monitor represents the distribution of
photon emission, and, accordingly, the location of light-emitting
conjugates.
[0182] In order to provide a frame of reference for the conjugates,
a greyscale image of the (still immobilized) subject from which
photon emission was measured is typically constructed. Such an
image may be constructed, for example, by opening a door to the
imaging chamber, or box, in dim room light, and measuring reflected
photons (typically for a fraction of the time it takes to measure
photon emission). The greyscale image may be constructed either
before measuring photon emission, or after.
[0183] The image of photon emission is typically superimposed on
the greyscale image to produce a composite image of photon emission
in relation to the subject.
[0184] If it desired to follow the localization and/or the signal
from a light-emitting conjugate over time, for example, to record
the effects of a treatment on the distribution and/or localization
of a selected biocompatible moiety, the measurement of photon
emission, or imaging can be repeated at selected time intervals to
construct a series of images. The intervals can be as short as
minutes, or as long as days or weeks.
[0185] F. Analysis of Photon Emission Images
[0186] Images generated by methods and/or using compositions of the
present invention may be analyzed by a variety of methods. They
range from a simple visual examination, mental evaluation and/or
printing of a hardcopy, to sophisticated digital image analysis.
Interpretation of the information obtained from an analysis depends
on the phenomenon under observation and the entity being used.
[0187] The following experiments illustrate one application of the
present invention--tracking Salmonella infection in live mice--and
how images obtained using methods of the present invention can be
analyzed. Similarly, infection of numerous other pathogens,
including, but not limited to, Pseudomonas, Staphylococcus,
Streptococcus, Enterococcus, Enterobacter, Citrobacter, Leginella,
Helicobacter, Acinetobacter, Escherichia, Klebsiella and
Serratia.
[0188] G. Imaging of Luminescent Salmonella in Living Mice
[0189] Experiments performed in support of the present invention
characterize the distribution of Salmonella typhimurium infection
in mice, the animal model of human typhoid. A mouse virulent
Salmonella typhimurium strain, SL1344 (Hoiseth and Stocker, 1981,
Nature 291:238-239), a non-invasive mutant of SL1344, BJ66 and a
low virulence LT-2 strain of Salmonella, LB5000 were each marked
with a plasmid containing the lux operon, and used in experiments
to localize Salmonella infection in mice.
[0190] 1. Constructions of Luminescent Salmonella
[0191] Salmonella Strains. Three strains of Salmonella typhimurium
with differing virulence phenotypes, defined by oral and
intra-peritoneal inoculations into mice, are selected for
transformation.
[0192] The most virulent phenotype used herein is SL1344, a mouse
strain originally obtained from a fatal infection of a calf
(Hoiseth and Stocker, 1981, Nature 291:238-239). Following oral
inoculations of mice with this strain, bacteria are disseminated
systematically via the lymphatic system resulting in colonization
of the liver, spleen and bone marrow (Carter and Collins, 1974, J.
Exper. Med. 139:1189-1203.; see also reviews by Finlay and Falkow,
1989, Mol. Microbiol. 3:1833-1841, and Hsu, 1989, Microbiol. Rev.
53:390-409.)
[0193] A non-invasive mutant of SL1344, BJ66, is also evaluated.
Systemic infections in mice do not typically result from an oral
inoculation with BJ66, but do result from intraperitoneal
inoculations with this strain.
[0194] A low virulence LT-2 strain of Salmonella, LB5000, is also
examined. LT-2 stains are laboratory strains known to be of reduced
or variable virulence for mice. LB5000 contains multiple
auxotrophic mutations, is streptomycin resistant, and is cleared
from mice following oral or intraperitoneal inoculations.
[0195] Transformation of Salmonella Strains with the lux Operon.
The three strains are each transformed with a plasmid encoding the
lux operon, as detailed in Example 1. The plasmid, obtained from
the soil bacterium Xenorhabdus luminescens (Frackman, et al., 1990)
confers on E. coli the ability to emit photons through the
expression of the two subunits of the heterodimeric luciferase and
three accessory proteins, luxC, luxD and luxE.
[0196] Inclusion of luxC, luxD and luxE removes the necessity of
providing the fatty aldehyde substrate, luciferin, to the
luciferase-expressing cells. Because supplying the substrate to
eukaryotic luciferase enzymes in an in vivo system such as
described herein may prove difficult, the entire lux operon of X.
luminescens is used. The operon also encodes the enzymes for the
biosynthesis of the fatty aldehyde substrate.
[0197] X. luminescens luciferase, an alpha-beta heterodimeric
mixed-function oxidase, catalyzes the oxidation of reduced flavin
and long-chain aldehyde to oxidized flavin and the corresponding
long-chain fatty acid. A fatty acid reductase complex is required
for the generation and recycling of fatty acid to aldehyde, and an
NAD(P)H:flavin oxidoreductase supplies the reduced flavin.
[0198] Optimal bioluminescence for E. Coli expressing the lux genes
of X. luminescens is 37.degree. C. (Szittner and Meighen, 1990, J.
Biol. Chem. 265:16581-16587, Xi, et al., 1991, J. Bact.
173:1399-1405). In contrast, luciferases from eukaryotic and other
prokaryotic luminescent organisms typically have lower temperature
optima (Campbell, 1988, Chemiluminescence. Principles and
Applications in Biology and Medicine (Chichester, England: Ellis
Horwood Ltd. and VCH Verlagsgesellschaft mbH)). The luciferase from
X. luminescens, therefore, is well-suited for use as a marker for
studies in animals.
[0199] The three strains are transformed by electroporation with
the plasmid pGSL1, which contains the entire X. luminescens lux
operon and confers resistance to ampicillin and carbenicillin on
the Salmonella (Frackman, et al., 1990). The X. luminescens lux
operon contains the genes luxA, luxB, luxC, luxD and luxE
(Frackman, et al., 1990). LuxA and B encode the two subunits of the
heterodimeric luciferase. luxC and D encode the biosynthetic
enzymes for the luciferase substrate and luxE is a regulatory gene.
Inclusion of the genes for the biosynthesis of the substrate is a
convenient means of providing substrate to luciferase, in contrast
to supplying luciferin externally to the cells in culture or
treating animals with the substrate.
[0200] 2. Characterization of Transformed Salmonella In Vitro
[0201] Adherence And Invasive Properties. The adherence and
invasive properties of the three Salmonella strains containing the
lux plasmid are compared in culture, to each other, and to their
non-luminescent parental strains by the standard invasion assay as
described by Finlay and Falkow, 1989, Mol. Microbiol. 3:1833-1841.,
and detailed in Example 2.
[0202] In this assay, adherent and intracellular bacteria are
quantified following incubation with an epithelial cell line and
peritoneal macrophages. The adherent and intracellular bacteria are
detected and quantified by both the emission of photons from living
cells, and colony forming units following lysis and plating the
cell lysates on carbenicillin-containing plates.
[0203] The results of some of the assays are shown in FIGS. 2A
through 2E and discussed in Example 8. The phenotypes of the three
strains transformed with the lux expressing plasmid are not
significantly altered in comparison to the parental Salmonella
strains. In addition, there is a good correlation between the
intensity of bioluminescence and the CFU from the HEp-2 cells and
macrophages. The results show that luminescence, as an indicator of
intracellular bacteria, is a rapid method for assaying the invasive
properties of bacteria in culture.
[0204] BJ66 demonstrated reduced adherence to HEp-2 cells in
comparison to SL1344, however, adherence of the two strains in
primary cultures of murine peritoneal macrophages were
comparable.
[0205] Light Emission. To evaluate the oxygen requirements of the
system, 10 fold serial dilutions of bacteria are placed in glass
capillary tubes and imaged, as detailed in Example 3.
[0206] FIG. 3 shows an image generated in one such experiment.
Luminescence is only detected at the air-liquid interface, even in
the tubes with small numbers of bacteria in air saturated medium
(0.1 ml of air saturated buffer in 5 l results in a final O.sub.2
concentration of 5 nM).
[0207] From these results, it is apparent that oxygen is likely a
limiting factor for luminescence.
[0208] Light Transmission Through Animal Tissue. To determine the
degree to which light penetrates animal tissue, light emitted from
luminescent Salmonella and transmitted through tissue is quantified
using a scintillation counter, with the fast coincidence detector
turned off to detect single photons. The background due to dark
current of the photomultiplier tubes in this type of detection is
significant, limiting the assay to samples with relatively strong
photon emission.
[0209] Four tissue types of varying opacity are compared using this
approach: muscle from chicken breast, skin from chicken breast,
lamb kidney and renal medulla from lamb kidney. The number of
photons that can be detected through tissue is approximately ten
fold less than the controls without tissue.
[0210] 3. Characterization of Lux Salmonella In Vivo
[0211] Oral Administration. Oral inoculation is natural route of
infection of mice or humans with Salmonella and results in a more
protracted course of disease. In order to study the progression of
the Salmonella infection following this route of inoculation, two
strains of mice are infected with the three strains of Salmonella.
The results obtained using the resistant animals are discussed
under the heading "Infection of Resistant Mice", below.
[0212] Balb/c mice are orally infected with suspensions of virulent
SL1344lux, non-invasive BJ66lux and low virulence LB5000lux
Salmonella, as described in Example 5. Progression of the infection
is followed by external imaging (Materials and Methods) over an 8
day period.
[0213] Representative images are shown in FIGS. 6A, 6B, and 6C. At
24 hours post inoculation (p.i.), the bioluminescent signal is
localized at a single focus in all infected animals (FIGS. 6A, 6B
and 6C). Bioluminescence disappears in all animals infected with
the low virulence LB5000lux by 7 days p.i. (FIG. 6A). Animals
infected with the virulent SL1344lux, on the other hand, show
virulent infection which often spreads over much of the abdominal
cavity (FIG. 6C), though the time at which it begins to spread is
highly variable from animal to animal. The infection by BJ66lux
typically persists and remains localized at a single site (FIG.
6B).
[0214] I.P. Inoculation. To assess whether or not there is
sufficient O.sub.2 at the sites of Salmonella replication for the
oxidation of luciferin and subsequent luminescence (Campbell, 1988,
Chemiluminescence. Principles and Applications in Biology and
Medicine (Chichester, England: Ellis Horwood Ltd. and VCH
Verlagsgesellschaft mbH)), photon emission is measured from the
tissues of a respiring animal. Luminescent SL1344lux and LB5000lux
are inoculated into the peritoneal cavities of two groups of Balb/c
mice. 32 hours post inoculation (p.i.), the transmitted photons are
imaged (FIG. 7).
[0215] In the mice infected with SL1344lux (left part of FIGURE),
transmitted photons are evident over a large surface, with foci of
varying intensities visible. These images are indicative of a
disseminated infection, and are consistent with widespread
colonization of the viscera, possibly including the liver and
mesenteric lymph nodes. In contrast, the distributions of
transmitted photons from animals infected with the LB5000lux strain
is very limited, indicating a limited infection.
[0216] The LB5000lux-infected mice remained healthy for several
weeks p.i., while the SL1344lux-infected mice were nearly moribund
and euthanized at 4 days p.i.
[0217] These experiments indicate that the level of O.sub.2 in the
blood and or tissues is adequate for bioluminescence of lux
luciferase expressed by Salmonella. Furthermore, the experiments
are consistent with the invasive nature of the virulent strain
SL1344 in comparison to the reduced virulent laboratory strain
LB5000.
[0218] Infection Of Resistant Mice. Mice which are heterozygous at
the Ity locus (Ity.sup.r/s) are resistant to systemic infections by
S. typhimurium (Plant and Glynn, 1976, J. Infect. Dis. 133:72-78).
This locus, also called Bcg (Gros, et al., 1981, J Immunol.
127:2417-2421) or Lsh (Bradley, 1977, Clin. and Exper. Immunol.
30:130-140), regulates the pathogenic processes of certain
intracellular pathogens, such as Mycobacterium lepraemurium
(Forget, et al., 1981, Infect. Immunol. 32:42-47), M. Bovis
(Skamene, et al., 1984, Immunogenet. 19:117-120, Skamene and
Pietrangeli, 1991, Nature 297:506-509) and M. intracelluare (Goto,
et al., 1989, Immunogenetics 30:218-221). An analogous genetic
control of resistance and susceptibility to intracellular pathogens
appears to be in humans as well (M. tuberculosis (Stead, 1992,
Annals of Intern. Med. 116:937-941, Stead, et al., et al., 1990,
New Eng. J. Med. 322:422-427) and M. leprae).
[0219] The Ity locus is located on mouse chromosome 1 with two
allelic forms, Ity.sup.r (resistant, dominant) and Ity.sup.s
(sensitive, recessive). The gene encoded at the Ity locus
apparently affects the ability of macrophages to disrupt the
internalized pathogens (reviewed by Blackwell, et al., 1991,
Immunol. Lett. 30:241-248 (1991); see also Skamene, et al., 1984,
Immunogenet. 19:117-120, Skamene and Pietrangeli, 1991, Nature
297:506-509) which in turn, affects the down stream function of the
proposed macrophage-mediated transport of pathogens to other sites
within the infected host. Balb\c mice are Ity.sup.s/s and 129 mice
are Ity.sup.r/r. The heterozygous Balb\c.times.129 mice
(Ity.sup.r/s) are used in experiments detailed herein.
[0220] Resistant 129.times.Balb/c (Ity.sup.r/s) viable mice are
infected by intragastric inoculation of 1.times.10.sup.7 SL1344lux
Salmonella as detailed in Example 7. The animals are imaged daily
for 8 days post injection (d.p.i.).
[0221] Results are shown in FIGS. 8A (day 1) and 8B (day 8). The
luminescence, detected by external imaging, is apparent at 24 h
p.i., and appeared to localized to a single site in all animals.
The luminescent signal is present throughout the study period (up
to 8 days p.i.). The intensity of the luminescence and the location
of the luminescent source is somewhat variable over time within a
mouse and also from mouse to mouse. The luminescent tissue in all
infected animals is the cecum (see below) and the variability in
localization, and possibly intensity, is most likely due fact that
internal organs of rodents are not tightly fixed in position.
[0222] The apparent limited infection observed in these animals
supports the interpretation that the Ity restriction blocks
macrophage transport. The persistence of this infection for 10
days, however, suggests that there is adherence to the intestine
mucosa and prolonged shedding of bacteria in the feces of these
animals, as evidenced by luminescent fecal pellets. These results
indicate that the luminescent phenotype of the Salmonella in vivo
is retained over an 8 day duration in Ity restricted animals and
that localization is possible following an oral inoculation.
[0223] Internal Imaging Following Oral Inoculation. In order to
further localize the luminescent signal in the abdominal cavity,
infected mice are imaged following laparotomy (Example 8). The
predominant disease manifestation in all of the animals infected by
the oral route is an enlarged cecum (FIGS. 9A, 9B, 9C). The
"external" image (FIG. 9A) illustrates a focal luminescence, which
is revealed in the post-laparotomy image (FIG. 9B) to be the
cecum.
[0224] Injection of air into the intestine confirms the presence of
bacteria in other regions of the digestive tract. Bacteria in the
colon and rectum are likely expressing luciferase, but low oxygen
concentrations are likely limiting light emission from these
sites.
[0225] The images obtained from oral inoculation studies indicate
that the luminescent signal, at 2 days p.i. and at 7 days p.i.,
localizes almost entirely to the cecum in each of the animals
(Popesko, et al., 1990, A Colour Atlas of Anatomy of Small
Laboratory Animals Vol. Two: Rat Mouse Hamster (London England:
Wolfe)) except those infected with LB5000lux. Luminescence is also
apparent in the colon in some animals. By 7 days p.i., no
luminescence is detectable in the LB5000lux-infected animals. The
CFU present in the organs of these mice are determined at 2 and 5 d
p.i.
[0226] In animals infected intragastrically with the invasive
strain, SL1344lux, the luminescence in the cecum appears early and
precedes a systemic infection. In contrast, infections with the
non-invasive BJ66lux strain result in a persistent luminescence
from the cecum that remains, in some animals, for the entire course
of the study (8 days). By 8 days p.i., luminescence is detected
over much of the abdominal surface, resembling the distribution of
photons following an i.p. inoculation, in the Sh1344lux infected
mice.
[0227] Infections with SL1344lux appear to become systemic, as
predicted, with progressively more photons being emitted from an
increasing surface area. Luminescence appears to localize over the
abdomen in infections with all strains with little detectable
luminescence from outside this area. A large number of transmitted
photons are localized as a single focus over the abdomen suggesting
that even though the infection may be systemic, the greatest amount
of replication may be in areas surrounding the intestine.
[0228] Localization of the luminescence over the cecum indicates
that not only are there large numbers of organisms in this region
of the intestine, but also suggests that the Salmonella associate
with cells of the mucosa such that they can obtain sufficient
oxygen for luminescence. Emission of photons from luciferase is
oxygen dependent and the expected oxygen levels in the lumen of the
cecum, or intestine in general, are below the levels required for
luminescence. The luciferase reaction is not expected to be
functional in the intestine unless the bacteria can obtain oxygen
from cells of the intestinal epithelium.
[0229] Thus, the systemic infection seems to be related to the
invasive phenotype and not to simply adherence to epithelial cells
of the intestine. These experiments implicate the cecum in some
role in the pathogenic process either in the carrier state or as a
site of dissemination.
[0230] Monitoring the progression of infections to different
tissues may greatly enhance the ability to understand these steps
in the pathogenic process, and enable the screening for compounds
effective to inhibit the pathogen at selected steps.
[0231] Internal Imaging Following I.P. Inoculation. Mice infected
intraperitoneally with SL1344lux are imaged before and after
laparotomy (Example 9). The results are shown in FIG. 10. The
images demonstrate luminescence over a majority of the abdomen with
multiple foci of transmitted photons. The cecum does not appear to
contain luminescent Salmonella. The results from these experiments
indicate that all strains of Salmonella have sufficient O.sub.2 to
be luminescent in the early phases of infection. However, entry of
Salmonella into cells of the mucosa and subsequent systemic
infection is likely limited to strains with the invasive phenotype,
since systemic infections at later time points are only apparent in
SL1344lux-infected mice.
[0232] Effects of Ciprofloxacin on Salmonella Infection.
[0233] Experiments, detailed in Example 10, are performed to
demonstrate that non-invasive imaging is useful for following the
response of an infection to drugs. Mice are orally inoculated with
SL1344lux and treated with 100 mg of ciprofloxacin, an antibiotic
effective against Salmonella infections. The mice are imaged at
selected time periods following treatment, and the extent of
infection is quantitated by measuring photon emission. Photon
emission in treated mice is compared to values before the
initiation of treatment, and to values from control mice that had
been infected, but not treated. Results from one such experiment
are shown in FIGS. 11A, 11B, 11C, 11D, and 11E and discussed in
Example 10. Infection is significantly reduced in mice treated with
the antibiotic, compared both to the levels of pathogen at time
zero in treated animals, and to levels of pathogen in control
animals throughout the treatment period.
[0234] Effects Of Carbenenicillin Selection. Ducluzeau, et al.,
1970, Zeut. Bakt. S313:533-548., demonstrated that treatment of
animals with antibiotics facilitated colonization of the cecum with
Salmonella. The mice in the present experiments are maintained on
an antibiotic regime of intramuscular injections of carbenicillin
for the purpose of selecting the Amp.sup.r Salmonella containing
the luciferase clone. This treatment may alter the course of the
gastrointestinal infection, but the observation that Salmonella can
associate with the cells lining the cecum indicates that oxygen is
available for luminescence. This observation is notable, since the
lumen of the cecum is commonly thought to be an anaerobic
environment.
[0235] H. Applications
[0236] The bioluminescence technology is broadly applicable to a
variety of hostpathogen systems and may also enable temporal and
spatial evaluation of other biological events, as for example tumor
progression and gene expression in living mammals, and have
application in pharmaceutical development and screening. Widespread
use of in vivo imaging of pathogens may reduce the numbers of
animals and time needed for experiments pertaining to pathogenesis
and/or the real-time study antimicrobial agents. Furthermore,
bioluminescent organisms may be useful as biosensors in the living
animal, much as luminescent bacteria are used in environmental
analyses. Korpela et al., for example, demonstrate that the limited
oxygen supply in the lumen of the G.I. tract restricted
bioluminescence to sites in which oxygen is accessible to the
Salmonella, perhaps directly from epithelial or other cell types.
Korpela, et al., 1989, J. Biolum. Chemilum. 4:551-554. This oxygen
requirement may find utility as an indicator of intimate cell-cell
interactions, or as a biosensor for studying oxygen concentrations
at various sites in living animals. In the following, several
exemplary applications of this technology are described for the
purpose of illustration, but are in no way intended to limit the
present invention.
[0237] 1. Determination of Oxygen Levels
[0238] The oxygen requirement for luminescence of luciferase
evidenced in the experiments summarized above indicates that the
present invention may be applicable as a method of determining
spatial gradients of oxygen concentration in a subject. Luminescent
bacteria have been used to measure oxygen levels in the range of
10-1 mM. The studies predict that 0.1 nM is the lower limit of
detection (Campbell, 1988, Chemiluminescence. Principles and
Applications in Biology and Medicine (Chichester, England: Ellis
Horwood Ltd. and VCH Verlagsgesellschaft mbH)). The imaging methods
described herein may be used for studying oxygen levels at various
sites in living animals. For example, microorganisms that have been
engineered to emit light in an O.sub.2 or Ca.sup.2+-dependent
manner could be used as biosensors in a subject, much like
luminescent bacteria are used in environmental analyses (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. Biolum.
Chemilum. 5:115-122). The dynamic range of luminescence with
respect to O.sub.2 concentration is much broader and reaches lower
O.sub.2 concentrations than O.sub.2 probes (Campbell, 1988,
Chemiluminescence. Principles and Applications in Biology and
Medicine (Chichester, England: Ellis Horwood Ltd. and VCH
Verlagsgesellschaft mbH)). Moreover, light emission in proportion
to O.sub.2 concentration is linear over a range of 30 nM to 8 mM,
and 9 mM O.sub.2 is required for 1/2 maximal luminescence.
[0239] 2. Localization of Tumor Cells
[0240] The growth and metastatic spread of tumors in a subject may
be monitored using methods and compositions of the present
invention. In particular, in cases where an individual is diagnosed
with a primary tumor, LECs directed against the cells of the tumor
can be used to both define the boundaries of the tumor, and to
determine whether cells from the primary tumor mass have migrated
and colonized distal sites.
[0241] For example, LECs, such as liposomes containing antibodies
directed against tumor antigens and loaded with LGMs, can be
administered to a subject, allowed to bind to tumor cells in the
subject, imaged, and the areas of photon emission can be correlated
with areas of tumor cells.
[0242] In a related aspect, images utilizing tumor-localizing LECs,
such as those described above, may be generated at selected time
intervals to monitor tumor growth, progression and metastasis in a
subject over time. Such monitoring may be useful to record results
of anti-tumor therapy, or as part of a screen of putative
therapeutic compounds useful in inhibiting tumor growth or
metastasis.
[0243] Alternatively, tumor cells can be transformed, transduced,
transiently or permanently, or otherwise made to emit light, with a
luciferase construct under the control of a constitutively-active
promoter, and used to induce luminescent tumors in animal models,
as described above. Such animal models can be used for evaluating
the effects of putative anti-tumor compounds.
[0244] 3. Localization of Inflammation
[0245] In an analogous manner to that described above, compositions
and methods of the present invention may be used to localize sites
of inflammation, monitor inflammation over time, and/or screen for
effective anti-inflammatory compounds. Molecules useful for
targeting to sites of inflammation include the ELAN family of
proteins, which bind to selections. An ELAN molecule can be
incorporated as a targeting moiety on an entity of the present
invention, and used to target inflammation sites.
[0246] Alternatively, an animal model for the study of putative
anti-inflammatory substances can be made by making the animal
transgenic for luciferase under the control of the E-selectin
promoter. Since E-selectin is expressed at sites of inflammation,
transgenic cells at sites of inflammation would express
luciferase.
[0247] The system can be used to screen for anti-inflammatory
substances. Inflammatory stimuli can be administered to control and
experimental animals, and the effects of putative anti-inflammatory
compounds evaluated by their effects on induced luminescence in
treated animals relative to control animals.
[0248] 4. Localization of Infection
[0249] As illustrated in experiments performed in support of the
present invention and summarized above, LGCs may be effectively
used to follow the course of infection of a subject by a pathogen,
including, but not limited to, Pseudomonas, Staphylococcus,
Streptococcus, Enterococcus, Enterobacter, Citrobacter, Leginella,
Helicobacter, Acinetobacter, Escherichia, Klebsiella or Serratia.
In experiments detailed herein, the LGCs are pathogenic cells
(Salmonella) transformed to express luciferase. Such a system is
ideally-suited to the study of infection, and the subsequent spread
of infection, in animal models of human diseases. It provides the
ability to monitor the progression of an infectious disease using
sites of infection and disease progression rather than traditional
systemic symptoms, such as fever, swelling, etc. in studies of
pathogenesis.
[0250] Use of an external imaging method to monitor the efficacy of
anti-infectives permits temporal and spatial evaluations in
individual living animals, thereby reducing the number of animals
needed for experiments pertaining to pathogenesis and/or the study
anti-infective agents.
[0251] 5. Monitoring Promoter Activity in Transgenic Mice
[0252] The generation of transgenic animals has become an important
tool in basic research and in the development of gene therapies and
gene vaccines. The present invention provides methods for rapid in
situ assessment of the uptake of nucleic acids and their expression
and thus the evaluation of gene delivery systems and DNA-based
therapies.
[0253] More specifically, luciferase expression may serve as a
real-time bioluminescent reporter, allowing the noninvasive
assessment of the level of promoter activity in living animals.
Photons from the in vivo luciferase reaction in the transgenic
animal are detected by a CCD camera, after transmission through
animal tissues, and used as an indication of the level and location
of gene expression. This way, a real-time assessment of the extent
of promoter activity in both superficial and deep tissues can be
accomplished.
[0254] As described in specific embodiments of the present
invention, the light-emitting reporter systems in transgenic
animals facilitate in vivo assessment of the regulation of gene
expression, thus facilitating the development of novel therapies
that target regulation of viral and host gene expression.
Bioluminescent reporters offer the advantages of spontaneous
emission of light without a need for outside light sources, low
background signal permitting near single-event detection, real-time
analyses, and the absence of cytotoxic photosensitizing dyes. As
such, bioluminescent reporters have a greater versatility than
fluorescent markers in mammalian tissues. Biological processes can
be viewed in vivo by illuminating the temporal and spatial
distribution of gene expression in animals and humans.
[0255] The in vivo monitoring of promoter activity as described
herein can be used for the assessment of gene delivery and
expression in gene therapies, gene vaccines, antisense
oligonucleotide therapies, the generation of chimeric and
transgenic animals in research. The technology is further useful
for real-time noninvasive assays for gene expression in research
environments involving questions of developmental regulation,
response to infectious disease or other systems where gene
expression demonstrates change.
[0256] The following examples are given to enable those skilled in
the art to more clearly understand and to practice the present
invention. The present invention is not limited in scope by the
exemplified embodiments, which are intended as illustrations of
single aspects of the invention only, and methods which are
functionally equivalent are within the scope of the invention.
Indeed, various modifications of the invention in addition to those
described herein will become apparent to those skilled in the art
from the foregoing description and accompanying drawings. Such
modifications are intended to fall within the scope of the appended
claims. The present invention is explained in more detail by means
of the below examples.
VI. EXAMPLES
A. Materials and Methods
[0257] 1. Cells
[0258] Salmonella strains SL1344 and LB5000 were obtained from B.
A. D. Stocker (Stanford University; Hoiseth and Stocker, 1981,
Nature 291:238-239). Salmonella strain BJ66 was obtained from B. D.
Jones (Stanford University).
[0259] HEp-2 cells were obtained from the American Type Culture
Collection (ATCC; 12301 Parklawn Dr., Rockville Md.; Accession
number CCL-23).
[0260] Murine peritoneal macrophages were obtained by peritoneal
lavage of euthanized Balb/c mice with 7 ml of growth medium
(Maximow and Bloom, 1931, Textbook of Histology, Saunders,
Philadelphia.)
[0261] 2. Static Cultures
[0262] Low oxygen (static) cultures were prepared by inoculating 3
ml of LB Broth containing 100 mg/ml of carbenicillin with 6 .mu.l
of a bacterial suspension from a stationary phase culture, and
growing the bacteria at 37.degree. C. overnight in a stationary 7
ml culture tube.
[0263] 3. Mice
[0264] Balb/c (Ity.sup.s/s) mice were obtained from the Department
of Oncology, Stanford University. 129.times.Balb/c (Ity.sup.r/s)
mice were obtained from the Stanford Transgenic Animal Facility
(Stanford, Calif.). All animals were housed under identical
conditions of photo period, feeding regime and temperature in the
Stanford University Research Animal Facility (Stanford,
Calif.).
[0265] Anesthesia was performed by injecting the animals
intraperitoneally (i.p.) with 33 .mu.g/kg body weight nembutal.
[0266] Euthanasia was performed by asphyxiation in CO.sub.2 or
cervical dislocation, following protocols recommended by the
Stanford University Research Animal Facility. Cervical dislocation
was used in experiments in which results may have beer, affected by
physiological changes due to asphyxia.
[0267] Mice infected with lux-transformed Salmonella were given
daily intramuscular (i.m.) injections of carbenicillin (125 mg per
kg body weight) to maintain selective pressure on the luminescent
Salmonella for retention of the Amp.sup.r plasmid containing the
lux operon.
[0268] 4. Imaging
[0269] Animals or objects to be imaged were immobilized in a
light-tight box containing a door and a charge-coupled device (CCD)
camera with a two stage microchannel intensifier head (model
C2400-40, Hamamatsu). The camera was attached, via cables leading
out of the box, to an "ARGUS 50" image processor (Hamamatsu).
[0270] The ICCD system described above is capable of detecting
single photons once a threshold of 10-30 photons is achieved. The
signal to noise ratio of the system ranged from 2:1 to
1.times.10.sup.4:l, depending on signal intensity.
[0271] Grey-scale images were obtained by opening the light box
door in dim room light and integrating for 8-64 frames. The gain
for the gray scale images was set to optimize the image--typically
at 3000 volts on a scale of 0 to 10,000 volts.
[0272] Bioluminescence data were obtained in absence of external
illumination. Exposure settings were as follows: the black level
was set automatically by the camera/image processor, the gain was
adjusted automatically by the intensifier controller, and the
f-stop was set at 2.8. A 60 mm "AF NIKKOR" macro lens was used
(Nikon Inc., Melville, N.Y.).
[0273] Bioluminescence images were generated by integrating photons
for a selected period of time, typically 5 minutes. Data are
presented at the lowest bit range setting of 0-3 bits per pixel for
all animals. For images of other objects, i.e., 24 well plates,
where the resolution of the bioluminescent signals was not possible
at a bit range of 0-0.3, the range was increased to a setting that
permitted localization of bioluminescent signals, typically 1-7.
Objects were imaged for shorter periods of time when additional
information could not be obtained by imaging for five minutes.
[0274] External imaging refers to non-invasive imaging of animals.
Internal imaging refers to imaging after a partial dissection of
the animals, typically a laparotomy. Internal imaging is performed
in selected animals to confirm the sources of photon emission
localized by external imaging.
[0275] The bioluminescence image data are presented as a
pseudo-color luminescence image representing the intensity of the
detected photons. Six levels of intensity are typically used,
ranging from blue (low intensity) to red (higher intensity).
[0276] To generate the FIGURES presented herein, greyscale and
bioluminescence images were superimposed, using the image
processor, to form a composite image providing a spatial frame of
reference.
[0277] The composite image was displayed on an RGB CRT (red, green,
blue; cathode ray tube) monitor, and the monitor was photographed
to produce hardcopies. Hardcopies were also generated by saving the
image processor image as a digital file, transferring the file to a
computer, and printing it on a color printer attached to the
computer. Alternatively, hardcopies may be generated by printing
the video signal directly using a video printer.
B. Example 1
Transformation of Salmonella with pCGLS1 lux Plasmid
[0278] Salmonella strains SL1344, BJ66 and LB5000 were transformed
with pCGLS1, a pUC18-based vector encoding the lux operon from
Xenorhabdus luminescens (Frackman, et al., 1990).
[0279] 1. pCGLS1 Plasmid
[0280] A schematic of the pCGLS1 plasmid is shown in FIGS. 1A, 1B
and 1C. The plasmid was constructed by cloning an -11 kb region
encoding the lux genes from the soil bacterium Xenorhabdus
luminescens (FIG. 1A; Frackman, et al., 1990) into the Bam HI site
(FIG. 1B) of pUC18 (FIG. 1C; Clontech, Palo Alto, Calif.). The
construction of the vector is described by Frackman, et al.,
(1990).
[0281] Restriction enzyme sites in FIG. 1A are represented as
follows: Bs, Bst EII; 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. A sequence
included in the multiple cloning site (MCS) is provided in FIG. 1B,
with the Bam HI site indicated in bold type.
[0282] A graphical representation of a pUC18 vector with no insert
is shown in FIG. 1C. Labeled elements include an ampicillin
resistance gene (Ap), a lac Z gene (lac Z) and an E. coli origin of
replication (Ori). The unmodified pUC18 vector is approximately 2.7
kb in size.
[0283] 2. Transformation of Salmonella
[0284] Electrocompetent cells from Salmonella strains SL1344, BJ66
and LB5000 were made using standard methods (Sambrook, et al.,
1989, In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Vol. 2) and stored at -80.degree. C. until just
prior to use. Electroporation was performed as follows: 1 .mu.l of
the plasmid (0.2 .mu.g/ml) was added to 40 .mu.l of ice-cold
electrocompetent cells suspended in 10% glycerol. The suspension
was mixed gently for one minute, placed in a 1 mm gap
electroporation cuvette and electroporated using a Bio-Rad
Gene-Pulser (Bio-Rad Laboratories, Hercules, Calif.). The settings
were 2.5 kvolts, 400 ohms and 25 .mu.farads.
[0285] Following a one hour agitated incubation in Luria Bertini
(LB) broth at 37.degree. C., the cells were plated on (LB) Agar
containing 100 .mu.g/ml carbenicillin and allowed to grow
overnight.
[0286] To maximize the bioluminescence of the labelled Salmonella,
the lux operon was maintained on a high-copy-number plasmid and not
integrated as a single copy gene.
[0287] However, plasmids are subject to modification by the
bacterial cell especially in recA strains, such as SL1344 and BJ66
used in this study. The recA locus encodes a recombinase that may
delete regions of the plasmid containing the lux operon and the
.beta.-lactamase. Therefore, Salmonella recovered from cells in
culture were plated both in the presence or absence of
carbenicillin, and were imaged to determine the frequency at which
bioluminescence was lost. All colonies recovered from
gentamicin-treated, lysed HEp-2 cells and macrophages were
ampicillin resistant (Amp.sup.r) and bioluminescent. Therefore, lux
genes appeared not to be lost during co-culture with mammalian
cells.
[0288] Colonies were assayed for luminescence by visual inspection
in a dark room. Five transformants were identified as having high
levels of luminescence. Three of these, one each from the SL1344,
BJ66 and LB5000 strains, were selected for subsequent experiments.
They were termed SL1344lux, BJ66lux and LB5000lux,
respectively.
C. Example 2
Invasive Potential of Normal and Transformed Salmonella
[0289] The invasive potential of six strains of Salmonella
(SL1344lux, LB5000lux, BJ66lux, SL1344, LB5000 and BJ66) was
determined using two types of bacterial adherence and entry assays.
Colony-forming units (CFU) assays were performed essentially as
previously described (Finlay and Falkow, 1989, Mol. Microbiol.
3:1833-1841) with modifications (Lee, et al., 1990, PNAS
87:4304-4308). Bioluminescence assays were performed essentially
like the CFU assays, except that the number of cells was
quantitated using bioluminescence, as opposed to CFUs.
[0290] Briefly, HEp-2 cells and primary murine peritoneal
macrophages were seeded into 24-well tissue culture dishes at
1.times.10.sup.5 cells per well in RPMI (Gibco/BRL, Grand Island,
N.Y.) supplemented with 20 mM glutamine (Gibco/BRL) and 5% fetal
calf serum (Hyclone, Logan, Utah). Twenty four hours (HEp-2) or
seven days (macrophages) after cell seeding, bacteria from static
cultures (see "Materials and Methods", above) were inoculated at
1.times.10.sup.6 (multiplicity of infection (m.o.i.) of 10) or
1.times.10.sup.7 (m.o.i. of 100, columns on right in FIGS. 2B, 2C,
2D, and 2E) organisms per well and centrifuged onto the cell
monolayer for 5 minutes at 1000 rpm (185.times.g) in a Beckman
clinical centrifuge (Beckman Instruments, Columbia, Md.). The
medium was replaced with RPMI medium (Gibco/BRL) either with (entry
assay) or without (adherence assay) gentamicin (100 mg/ml). The
co-cultures were incubated for a total of 3.5 hours at 35.degree.
C. in 5% CO.sub.2.
[0291] Gentamicin in the incubation medium kills bacteria that had
not been internalized by the HEp-2 cells, including those adhering
to the surfaces of the HEp-2 cells. Accordingly, the signal in
adherence assays (without gentamicin) represent both adherent and
internalized bacteria, whereas the signal in entry assays (with
gentamicin) represent only internalized bacteria.
[0292] Adherence and entry were assayed by imaging luminescent
bacterial cells at three timepoints--1.5, 3.0 and 3.5 hours post
inoculation. Prior to imaging at the first timepoint, the cell
monolayer was washed three times with phosphate-buffered saline
(PBS) to remove unattached bacteria and a fresh aliquot of RPMI
medium was added. Luminescence was recorded using a 30 second
exposure. Images at the second and third timepoints were obtained
using a similar exposure, but without first washing the cells.
[0293] Data recorded at the last timepoint, displayed as
pseudocolor luminescence images superimposed over gray scale images
of the culture dish wells, are shown in FIG. 2A. The cell types,
Salmonella strains, and usage of gentamicin are indicated in the
FIGURE. The data are also summarized as relative intensity of
photon counts in the graphs in FIGS. 2B and 2D.
[0294] Following imaging at the 3.5 hour timepoint, the tissue
culture cells were washed three times with PBS and lysed with 0.2%
"TRITON X-100" in PBS. Adherent and/or intracellular bacteria,
released by lysis, were plated on LB- or LB-carbenicillin agar
plates and incubated for 18 h at 35.degree. C. The number of
bacteria released from each well was determined by counting the
number of colony forming units (CFU, Finlay and Falkow, 1989, Mol.
Microbiol. 3:1833-1841., Lee, et al., 1990, PNAS 87:4304-4308).
These data are represented as the total bacterial colonies per ml
recovered from co-culture after incubation for 3.5 h with or
without gentamicin, and are summarized in the graphs in FIGS. 2C
and 2E.
[0295] Data from both the bioluminescence and CFU assays indicate
that (i) Salmonella transformed with the lux genes have an
infective potential similar to that of the parent lines, and (ii)
luminescence detection and CFU determination yield comparable
estimates for the invasive potential of the two Salmonella strains
in HEp-2 cells and macrophages. The ratio of bioluminescence to CFU
was lower in macrophage cultures, possibly due to the subcellular
compartment in which the Salmonella enter macrophages.
D. Example 3
In Vitro Luminescence of Transformed Salmonella
[0296] 10 .mu.l of four 10-fold serial dilutions (ranging from
10.sup.6 cells to 10.sup.3 cells per ml) of LB5000lux Salmonella
were placed in four 100 .mu.l glass capillary tubes (Clay-Adams
div. of Becton Dickinson, Parsippany, N.J.). The bacterial
suspensions formed columns of fluid in the tubes, with pockets of
air at both ends. One end of each tube was sealed with critoseal
(Clay-Adams). The medium in which dilutions were made was saturated
with O.sub.2 through exposure to air.
[0297] The tubes were wrapped with clear plastic wrap and
luminescence was determined by imaging for 30 seconds as described
above. An exemplary image is shown in FIG. 3A. Four tubes are
pictured. They contained (from top to bottom) 10.sup.6, 10.sup.5,
10.sup.4 and 10.sup.3 Salmonella cells/ml (10.sup.4, 10.sup.3,
10.sup.2 and 10 cells/tube). Luminescence could be detected in
suspensions containing as few as 10.sup.4 cells/ml (100 cells). The
luminescence is confined, however, to air/liquid interfaces,
suggesting that the luminescence reaction requires relatively high
levels of oxygen. Since many of the cells are presumably in the
fluid column and not at the air/fluid interfaces, the data suggest
that the luminescence in the capillary tubes shown in FIG. 3A
arises from considerably fewer than the total number of cells in
each tube.
E. Example 4
In vitro Detection of Luminescence through Animal Tissue
[0298] Micro test-tubes, constructed from glass capillary tubing
with an internal diameter of 3.5 mm, containing serial dilutions of
LB5000lux Salmonella were prepared essentially as described in
Example 3, above. In the present example, however, the bacterial
suspensions contacted the sealed end of the tube and were exposed
to air only at the upper end. The tubes were placed in a
translucent plastic scintillation vial and surrounded by one of the
following animal tissues: chicken breast muscle, chicken skin, lamb
kidney or lamb renal medulla. All tissues were obtained from the
meat department of a local supermarket (Safeway, Mountain View,
Calif.).
[0299] A diagram of a vial containing a capillary tube surrounded
by tissue is shown in FIG. 5. The vial 1 is approximately 1.4 cm in
diameter and includes a cap 2. The vial is coated with an opaque
material (i.e., black tape) along its upper portion 3. Animal
tissue 4 is placed in the vial such that it extends from the bottom
of the vial to just above the bottom edge of the opaque coating 3.
The micro test-tube 5 is sealed at the bottom by a plug 7 (i.e., a
crytoseal plug), and is centered radially in the vial, with the
plugged end of the tube touching or in close proximity to the
bottom of the vial. The bacterial suspension 6 extends
approximately 1 cm upward from the bottom of the tube.
[0300] Photons emitted from vials with and without tissue, and with
and without bacteria, were counted using a liquid scintillation
counter (model 1219 Rackbeta, LKB/Wallac, Gaithersburg, Md.) with
the fast coincidence discriminator disabled.
[0301] Controls without tissue were assayed by placing the
bacterial suspension directly in the scintillation vial. All
experiments were performed in triplicate.
[0302] In each experiment, the vials were counted two to three
times, rotating the vial 90.degree. between each count, to control
for effects of possible tissue thickness inconsistency. No
significant differences were detected.
[0303] The results are summarized in TABLE I, below.
TABLE-US-00001 TABLE I TRANSMISSION OF PHOTONS THROUGH TISSUE
Chicken Chicken Lamb Lamb Sample skin muscle kidney medulla Vial
alone 2.1 .times. 10.sup.4 1.3 .times. 10.sup.4 1.0 .times.
10.sup.4 1.0 .times. 10.sup.4 Tissue alone N.D. 1.5 .times.
10.sup.4 9.4 .times. 10.sup.3 8.5 .times. 10.sup.3 Tissue and 2.7
.times. 10.sup.5 2.3 .times. 10.sup.5 1.6 .times. 10.sup.4 1.5
.times. 10.sup.5 LB5000lux* LB5000lux* 2.0 .times. 10.sup.6 1.7
.times. 10.sup.6 4.8 .times. 10.sup.6 4.8 .times. 10.sup.6 alone
Counts are averages of triplicate measurements, tissue path length
was 1 cm. *1 .times. 10.sup.7 cells.
[0304] The signal for 1.times.10.sup.3 LB5000lux in kidney tissue
was at or near background levels using the photomultiplier tubes
(PMT) in the scintillation counter. The background in this type of
detection is due to the dark current of the PMT and limits the
studies to analysis of rather intense signals.
[0305] Bioluminescence from approximately 1.times.10.sup.7
LB5000lux was detectable through 0.5 cm of avian muscle, skin ovine
renal medulla and ovine kidney. These results indicate that
bioluminescence from the labeled Salmonella was detectable through
animal tissues of variable opacity. Since oxygen was likely limited
in the capillary tubes (as demonstrated in FIG. 3A), it is likely
that fewer numbers of bioluminescent Salmonella could be detected
through tissue than are indicated in this assay.
F. Example 5
In Vivo Detection of Bioluminescent Salmonella
[0306] To assess the availability of oxygen to Salmonella during
infection, wild-type SL1344lux was inoculated into the peritoneal
cavity (i.p.) of BALB/c mice. Photons emitted from the bacteria
internally, and transmitted through the abdominal wall were
externally detected and localized in anaesthetized mice using an
intensified CCD camera 24 h after inoculation (FIG. 3B). Systematic
Salmonella infections are thought to involve colonization of the
lymph nodes, spleen, liver. Ventral images of the mice infected by
i.p. inoculation of wild-type SL 1344lux demonstrated transmitted
photons over much of the abdominal surface, with foci of various
intensities (FIG. 3B). These results were consistent with
widespread colonization of the viscera, possibly including the
liver and mesenteric lymph nodes, and indicate that the level of
available oxygen in some tissues can be adequate foe external
detection of bioluminescence from the labelled pathogen.
G. Example 6
Effect of Human Blood on the Light Emission from Bioluminescent
Salmonella
[0307] As demonstrated in the following example, fewer than ten
(10) bacterial cells can be detected with an intensified CCD
detector.
[0308] Two fold serial dilutions of Salmonella, strain LB5000, that
had been transformed with a plasmid that conferred constitutive
expression of the luciferase operon were plated in duplicate into
96 well plates. Dilutions were made in 30 .mu.l of growth medium
alone (indicated as LB5000) and with 30 .mu.l of blood to determine
the effects of blood as a scattering and absorbing medium on the
limits of detection. Each dilution and the numbers of colony
forming units (CFU) implied from plating samples from concentrated
wells are indicated in FIG. 4. The relative bioluminescence for
each well as determined by analysis of the image generated by the
CCD detector is shown (FIG. 4). The signal in the more concentrated
wells was off scale and the numbers are therefore not linear at
higher concentrations.
H. Example 7
Detection of Orally-Administered lux Salmonella in Balb/c Mice
[0309] Balb/c mice were infected by oral feeding (Stocker, et al.)
with a 50 .mu.l suspension of 1.times.10.sup.7 virulent SL1344lux,
non-invasive BJ66lux and low virulence LB5000lux Salmonella. The
mice, 4-6 weeks of age at the time of infection, were imaged daily
with 5 minute integration times (photon emission was measured for 5
minutes). Prior to imaging, the mice were anesthetized with 33
.mu.g/kg body weight nembutal.
[0310] Representative images are shown in FIGS. 6A, 6B, and 6C. At
24 hours post inoculation (p.i.), the bioluminescent signal
localized to a single focus in all infected animals (FIGS. 6A, 6B,
and 6C). Bioluminescence disappeared in all animals infected with
the low virulence LB5000lux by 7 days p.i. (FIG. 6A). In BALB/c
mice infected with the wild-type SL1344lux, bioluminescence was
detected throughout the study period, with multiple foci of
transmitted photons at 8 d. In these animals, the infection
frequently spread over much of the abdominal cavity (FIG. 6C). In
one-third of these animals, transmitted photons were apparent over
much of the abdominal area at 8 d, resembling the distribution of
photons following an i.p. inoculation (see FIGS. 3B and 6F). The
spread of infection by BJ66lux was more variable, but the infection
typically persisted and remained localized at the initial site
(FIG. 6B).
[0311] After infection of resistant BALB/c.times.129 mice with
wild-type SL 1344lux, the bioluminescent signal remained localized
and persistent in a group of 10 mice throughout the study period.
This result was in contrast to the disseminated bioluminescence
observed in SL1344lux-infected susceptible mice (lty.sup.r/s) (see,
Example 9 and FIGS. 8A and 8B), but resembled the persistent
infection of susceptible BALB/c mice with the less invasive
BJ66lux. As a control, Salmonella were cultured from persistently
infected resistant BALB/c.times.129 mice, and 80-90% of the
colonies recovered after 8 d were Amp.sup.r. Of these, more than
90% were bioluminescent, suggesting that observed differences were
not due to significant loss of lux plasmid, but rather were due to
real differences in pathogenicity of the bacterial strains.
I. Example 8
Detection of Infection Following I.P. Inoculation with a Virulent
and a Low Virulence Strain of Salmonella
[0312] Balb/c mice were infected with either virulent (SL1344lux)
or low virulence (LB5000lux) Salmonella by intraperitoneal (i.p.)
inoculations of 1.times.10.sup.7 bacterial cells in a 100 .mu.l
suspension, without simultaneous injection of air.
[0313] At 32 hours post injection (p.i.), the mice were
anesthetized and imaged as described above. The results are shown
in FIG. 7. Widespread infection is evident in the two mice in the
left part of FIG. 7, infected with the virulent SL1344lux strain.
In contrast, little, if any, luminescence is detected in the mice
on the right, injected with the low virulence LB5000lux strain.
J. Example 9
Detection of Systemic Infection in Resistant Mice Following Oral
Inoculation with Salmonella
[0314] Resistant 129.times.Balb/c (Ity.sup.r/s) viable mice were
infected by intragastric inoculation of 1.times.10.sup.7 SL1344lux
Salmonella. The bacteria were introduced through an intra-gastric
feeding tube while under anesthesia. The animals were imaged daily
for 8 days post injection (d.p.i.).
[0315] Results are shown in FIGS. 8A and 8B. Mice, in triplicate,
were infected and imaged daily for 8 days. Exemplary images for day
1 (FIG. 8A) and day 8 (FIG. 8B) are shown. These data indicate that
mice resistant to systemic Salmonella infection have a localized
chronic infection in the cecum, but that the infection does not
spread into the abdominal cavity.
K. Example 10
Post-Laparotomy Imaging following Oral Inoculation with
Salmonella
[0316] Laparotomy was performed following oral inoculation of
Salmonella to precisely localize the luminescent signal within the
abdominal cavity, and to compare this localization with that
obtained using non-invasive imaging. The animals were inoculated as
described in Example 9. After a selected period of time, typically
seven days, the mice were anesthetized and externally-imaged, as
described above. An exemplary image is shown in FIG. 9A. After
external imaging, the peritoneal cavity was opened and the animals
were imaged again, as illustrated in FIG. 9B. In some instances the
mice were imaged a third time, following injection of air into the
lumen of the intestine both anterior and posterior to the cecum (C)
(FIG. 9C). The mice were euthanized immediately after the final
imaging.
[0317] In each case where a focal pattern of bioluminescence was
observed in susceptible mice, early in infection after oral
inoculation, photons originated almost exclusively from the cecum,
while variations in the precise localization and intensity of focal
bioluminescence were due to variable positioning of the cecum. The
focal pattern of bioluminescence observed in infection-resistant
BALB/c.times.129 mice similarly localized to the cecum. In
contrast, such localization was not observed in animals infected
i.p. with SL1344lux (FIG. 3B). At late stages in
infection-susceptible mice inoculated orally with the wild-type
SL1344lux, bioluminescence was multifocal, however, additional foci
of luminescence did not become apparent after laparotomy. In mice
infected with the less-virulent LB5000lux, bioluminescence was not
detectable at 7 d in any tissue or organ, even focally, after
removal of the skin and peritoneal wall.
[0318] Bioluminescence was not detected optically in the spleen or
bloodstream of any infected animal; bioluminescence from the liver
was seen only at later stages of disease; and bioluminescence from
the G.I. tract was restricted to the cecum early in the disease
course. This pattern could be due to differences in the Aumbers of
Salmonella in the different tissues, or lack of available oxygen.
The Amp.sup.r cfu present in homogenized organs of orally infected
mice were quantified to evaluate the distribution of labelled
Salmonella SL1344lux. Greater than 90% of the amp.sup.r bacterial
colonies obtained from all analyzed tissues of SL1344lux-infected
BALB/c mice at 7 d indicated total cfu from the liver, spleen, and
lungs were in the range of 1.9.times.10.sup.3 to
>1.0.times.10.sup.5 without detectable photon emission, in vivo
(TABLE II). In contrast, bioluminescence was detectable from the
cecum and this tissue contained >1.0.times.10.sup.8 total cfu.
No cfu were detectable in any tissue of the LB5000lux infected
mice. These results suggest that 1.times.10.sup.5 organisms in
tissue is near the limit of detection at this emission wavelength
using the current experimental system.
[0319] Oxygen is an essential substrate for the luciferase
reaction, thus only Salmonella present in oxygenated
microenvironments should be bioluminescent. The absence of
bioluminescence from Salmonella in the anaerobic environment of the
lumen of the G.I. tract is therefore predictable, and exposure of
the intestinal lumen to air should reveal the presence of bacteria
previously not detectable due to a lack of oxygen. In support of
this view, one animal with detectable bioluminescence in the cecum
alone excreted a faecal pellet that rapidly became bioluminescent
upon exposure to air. This indication of non-luminescent,
luciferase-expressing bacteria in the lumen of the intestine and
the clear delineation of the aerobic and anaerobic zones in this
tissue, suggested that injection of air into the lumen of the
intestine would reveal the presence of additional bacteria.
Injection of air into the lumen of the ileum and colon of another
animal, with a similar pattern of bioluminescence, resulted in
detectable photons near the injection sites (FIG. 9). Last, when a
third mouse with cecal bioluminescence was killed, bioluminescence
quickly ceased. Air was injected at other tissue sites because of
the lack of clear zones of aerobic and anaerobic environments.
TABLE-US-00002 TABLE II Colony-forming units in homogenized tissue
from mice infected with bioluminescent Salmonella Tissue Animal
Weight Total Strain Tissue Number (mg) cfu SL1344lux Liver 1 441
1.9 .times. 10.sup.3 2 778 2.5 .times. 10.sup.4 Spleen 1 218 1.2
.times. 10.sup.4 2 248 4.9 .times. 10.sup.5 Mesenteric 1 76 >1.0
.times. 10.sup.6 lymph node 2 46 >1.0 .times. 10.sup.6 Lung 1 17
1.5 .times. 10.sup.3 2 69 2.7 .times. 10.sup.3 Cecum 1 351 >1.0
.times. 10.sup.8* 2 422 >1.0 .times. 10.sup.8* *Photons emitted
from bacteria at these tissue sites were externally detected.
L. Example 11
Post-Laparotomy Imaging Following I.P. Inoculation with
Salmonella
[0320] Balb/c mice were infected by intraperitoneal inoculation of
1.times.10.sup.7 Salmonella (SL1344lux) as described in Example 8.
Exemplary images of one such animal are shown in FIGS. 10A, 10B and
10C.
[0321] At 24 hours post-injection (p.i.), the animal was
anesthetized and imaged for five minutes (FIG. 10A). The peritoneal
cavity was opened and the mouse was imaged again for five minutes
(FIG. 10B). The cecum was pulled to the left side, and the animal
was again imaged for five minutes (FIG. 10A).
[0322] The results demonstrate that the localization of infection
sites obtained with non-invasive imaging correlates well with the
sites as revealed upon opening the peritoneal cavity.
M. Example 12
Effects of Ciprofloxacin Treatment on Bioluminescence from
SL1344lux Salmonella
[0323] To demonstrate the utility of in vivo imaging, an infected
animal was treated with the antibiotic ciprofloxacin, which known
to be effective against systemic Salmonella infections. Magalianes,
et al., 1993, Antimicrobial Agents Chemo. 37:2293.
[0324] Experimental and control groups of Balb/c mice were orally
inoculated with SL1344lux. At 8 days p.i., mice in the experimental
group were injected i.p. with 100 mg of ciprofloxacin hydrochloride
(3 mg/kg body weight; Sigma Chemical Co., St. Louis, Mo.).
Following treatment of the experimental group, animals from both
groups were imaged (as above) at several intervals over a period of
5.5 h post treatment.
[0325] Representative images are shown in FIGS. 11B, 11C, 11D, and
11E. FIGS. 11B and 11D show composite images of representative
animals from the control and treated groups, respectively,
immediately before initiation of treatment of the experimental
group. FIGS. 11C and 11E show composite images of the same animals
5.5 hours after initiation of treatment. Bioluminescence over the
abdomen of the ciprofloxacin-treated animal was reduced to
undetectable levels during this period of time, while
bioluminescence in the control typically increased 7.5-fold. The
total number of photons detected over the abdominal area were
determined, normalized to the value at t=0, and plotted in FIG. 11A
with respect to time post-treatment.
[0326] The data demonstrate that methods and compositions of the
present invention can be used to evaluate the effects of drugs on
the spread of infection in vivo.
N. Example 13
Bioluminescent Reporter for Promoter Activity in Cultured Cells
[0327] In order to demonstrate how the promoter from HIV (human
immunodeficiency virus) responds to viral infection over time,
jurket cells transfected with a plasmid containing the HIV LTR
(long terminal repeat, promoter) upstream of the coding sequence of
firefly luciferase were infected with a laboratory isolate of HIV-1
(strain A111) using standard laboratory conditions and followed for
a period of 7 days (d) for emission of bioluminescent light. After
24 h, 60 h, 96 h, and 7 d, a gray scale image of the plate was
generated in low room light followed by collection of photons
emitted from the cultured cells in complete darkness for a period
of 10 min. A color pseudoimage representing the intensity of
bioluminescent light was superimposed over the gray scale image of
the plate (FIG. 12). At 7 d post infection a clear signal is
present in the duplicate cultures indicating high levels of
replication (FIG. 12). The images at different time points
represent the same two wells. Advantages of this assay for HIV
replication are that: i) temporal studies can be done in a minimum
number of wells since the same wells are followed over time, ii)
kinetics of replication can therefore be studied as a phenotypic
characteristic of viral isolates, iii) samples of cells or
supernatant do not have to be collected, iv) the level of viral
replication is almost immediately apparent, v) the detection could
be set up remotely limiting human handling, vi) antiviral drugs
could be evaluated in culture with the above listed advantages.
O. Example 14
Assessing Promoter Activity in Tissues of Transgenic Mice
[0328] Transgenic mice containing a construct composed of the
regulatory portion of the HIV LTR (U3 region) upstream of the
coding sequence of the firefly luciferase gene were generated and
evaluated for the emission of photons after transmission through
tissues. A diagram of the construct is shown at the bottom of FIG.
13. Numbers along the construct indicate nucleotide positions
relative to the start of transcription. Sequences matching known
motifs of cellular transcription factors are indicated with the
names of the factors preview of these factors can be found in ref.
[chris, where?]). Transcription from the HIV LTR was activated in
the right ear of each two animals with a single topical application
of dimethyl sulfoxide (DMSO). The animal on the left in FIG. 13 was
given 150 .mu.l of an aqueous solution of the substrate luciferin
(50 mg/ml) via intraperitoneal (i.p.) injection. The animal on the
right was not given substrate. 20 min. post treatment with
substrate the animals were imaged as described for the plate in
FIG. 12 with a 20 min. integration time. The color pseudoimage
indicates light emission over the right ear (B) of the animal on
the left, and not from the uninduced ear (A) or the animal that was
not given substrate (C,D). This is the first demonstration of
monitoring promoter activity in a living adult animal and
demonstrates the relatively tight regulation of the LTR with DMSO
induction. This technology allows for the temporal and spatial
analyses of transcriptional activity in living animals.
P. Example 15
Topical Delivery of Substrate to Dermal Cells in Transgenic
Animals
[0329] In order to optimize delivery of substrate, the substrate
was topically delivered to dermal cells. The HIV LTR was induced in
the skin of mice with twice daily treatments of DMSO over the
entire surface of the back and the right ear for two consecutive
days. Substrate was applied to the skin in solutions prepared in
DMSO. Concentrations included 200 mM, 100 mM, 50 mM, 25 mM, and
12.5 mM. 5 .mu.l of each concentration were spotted, in
quadruplicate, on the backs using a multichannel pipette with the
highest concentrations near the head. 5 .mu.l of the 50 mM solution
was applied to each ear. 2 min. after application the animal was
imaged as described in FIG. 12. The bioluminescent response
appeared increase linearly over the concentrations from 12.5 mM to
100 mM (FIG. 14). Bioluminescence from spots containing 200 mM
luciferin was roughly equivalent to that from the 100 mM spots,
solutions of luciferin containing water, in contrast, resulted in
no detectable bioluminescence (FIG. 14). Solutions of 25% H.sub.20
in DMSO to 100% H.sub.20 were tested.
Q. Example 16
Induction of Bioluminescence in Ears of Transgenic Animals by
Topical Luciferin Delivery
[0330] The experiment of induction of luciferase expression in ears
and systemic luciferin delivery of Example 14 was repeated with
topical administration of substrate in 100% DMSO (FIG. 15). Signals
from the ears were uniform and had greater intensity than with
systemic luciferin delivery. See, FIGS. 13 and 15. Peak light
emission was observed immediately after topical treatment compared
to 20-30 min. after systemic administration of substrate.
R. Example 17
Unilateral Induction of Luciferase Expression in Transgenic
Mice
[0331] The left half of the shaved dorsal surface of the transgenic
animals and the left ear were treated twice daily for two days with
DMSO to activate expression of the HIV-1 LTR. Luciferin was applied
topically over the entire surface of the back and both ears, and
animals were imaged immediately after addition of substrate.
Unilateral emission of bioluminescence corresponding to the induced
region was observed (FIG. 16).
S. Example 18
Bioluminescence Detectable in Internal Tissues of Transgenic
Animals
[0332] Bioluminescence was detectable from the abdomens of animals
treated with DMSO on one ear only. This signal is assumed to be due
to ingestion of DMSO during grooming (FIG. 17).
T. Example 19
Localization of Internal Bioluminescence in Transgenic Mice
[0333] Animals demonstrating signal from the abdomen were
laprotomized and imaged. Bioluminescent signal localized to the
colon in 4 of 4 animals studied and in the animals shown in FIG. 18
was tightly localized to a region of the colon about 1 cm in
length. In the other animals the entire colon appeared to emit
bioluminescent light.
U. Example 20
Expression of The HIV-LTR in Neonatal Transgenic Mice
[0334] As the demonstrated in the following experiment, the HIV-LTR
is differentially expressed through development.
[0335] 4 d old transgenic mice were given intraperitoneal
injections of luciferin in aqueous solution (15 .mu.l at 50 mM),
and imaged with integration times of 20 min. In the absence of any
known inducing agent or treatment, bioluminescent signal indicative
of expression of luciferase form the HIV LTR was apparent as a
diffuse signal over much of the surface of the animal with more
intense signal originating from the developing eye and extremities
(FIG. 19). These data demonstrate that the LTR is inherently active
in neonatal transgenic mice, and may be expressed to a greater
level in the eyes and other locations.
[0336] All references are hereby incorporated in their entirety.
Sequence CWU 1
1
1159DNAArtificial Sequencelux pCGLS1 plasmid polylinker sequence
1ccaagcttgc atgcctgcag gtcgactcta gaggatcccc gggtaccgag ctcgaattc
59
* * * * *