ivis - in vivo imaging system - jagiellonian...

IVIS - in vivo imaging system

Animal Facility of our Faculty

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Część techniczna:”brudna”, usuwanie ściółki, mycie klatek, sterylizacja

Część czysta:Zwierzęta rozrodcze, bez eksperymentów, dostęp ograniczony wyłącznie dla pracowników zwierzętarni

Część doświadczalna:Zwierzęta w doświadczeniu, lub czekające na doświadczenie, dostęp dla wszystkich eksperymentatorów

Śluzy

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Uwaga: zlewy/umywalki konieczne są tylko w trzech pomieszczeniach (2, 10, 11); kratka ściekowa tylko w pomieszczeniu 2. Czujniki ruchu – korytarz oraz pomieszczenie 2, 3, 4, 7, 10, 11.Regulacja fotoperiodu: pomieszczenia 5, 6, 8, 9, 12, 13, 14.

Major steps in a new drug development

Optical imaging

cyclotron

radioactivity

radioactivity

magnetic label

Advantages of IVIS:- High throughput- No radiation- Easy to operation- Dual function (fluorescence and bioluminescence)- Functional analysis

Optical imaging: IVIS

Non-invasivein vivo imaging- Continued monitoring- Low variation- Each animal can be used as its own control (averaging effects are eliminated) - 3R (Replacement, Reduction, Refinement)

Bioluminescence - production and emission of light by a living organism as the result of a chemical reaction during which chemical energy is converted to light energy.

Fluorescence - re-emission of the light absorbed from an exogenous source as another wavelength of light.

There is no exogenous light source with bioluminescence -no autofluorescence. This results in higher sensitivity detection compared to fluorescence.

Bioluminescence

Because it is a chemical reaction, the substrate must be injected just prior to each imaging session and light production will only last as long as the reaction continues (approximately 1-30 min depending on amount of substrate injected).

The source of light

- plasmids

- adenoviral vectors

- retroviral vectors

- lentiviral vactors

Transient or stable transfection with luciferase gene

560 nm

475 nm

Firefly luciferase and Renilla luciferase

Luciferin Oxyluciferin

Coelentrazine Coelentramide

Both luciferase proteins are monomers that do not require any posttranslational modifications; they are valiable as a mature enzyme directly upon translation from their respective mRNAs.

98% of the energy released in this oxidation-reduction reaction is in the form of light. The remaining 2% dissipates as heat.

Firefly luciferase is a 62 kDa monomer and Renilla luciferase is a 36 kDa monomer. The kinetics of these two enzymes varies significantly. The Renilla luciferase reaction will occur more rapidly than the Firefly luciferase reaction.

Firefly luciferase also requires ATP and magnesium. Because ATP is crucial for cell viability, therefore, when ATP is a potential variable, Renilla luciferase would be the suggested option for experimentation.


IVIS Lumina

IVIS Lumina

gas anaestesia system

Luc-transgenic mouse expresses luciferasein all organs

Janet Sawicki, Lakenau Institute for Medical Research

The IVIS® Lumina can be used to image fluorescent and bioluminescent reporters both in vitro and in vivo. Living Image software can be applied.

Serial dilutions of PC3M-luc prostatecancer cells (from 1x105 to 50 cells)

Subcutaneous tumor and metastasisof B16(F10) murine melanoma cells

IVIS-Lumina

IVIS Lumina

Bioluminescence and fluorescence

Fluorescent signal is limited by tissue autofluorescence- signal to background is 400x higher for bioluminescece

Tissue is not transparent - light absorbance depends on wavelength

Living Image software (for bioluminescent (DLIT4) and fluorescent (FLIT4) sources), which allows for:

Living image software

- signal location in anatomicalcontext,

- 3D visualization,

- quantitation in terms of cellnumber of cells expressing bioluminescent or fluorescent protein or pmols of a fluores-cent compound.

Dylite 800 (blue) - lungs

AF650 (red) - liver and bladder

Chlorophyll (yellow) - intestine

The IVIS 200, combines the ability to image in vitro or in vivo, fluorescence or bioluminescence, and 2D or 3D, in a single integrated imaging system.

It provides high resolution with "a single cell"sensitivity for in vitro or ex vivo use.

A laser scanning device provides the 3D surface topography used in diffuse tomographicreconstructions of internal sources.

IVIS 200

IVIS 200

35 µµµµm

IVIS 200

IVIS Spectrum

The IVIS Spectrum, combines the ability to image in vitro or in vivo, fluorescence or bioluminescence in2D or 3D.

It provides high resolution with "a single cell" sensitivity for in vitro or ex vivo use.

A laser scanning device provides the 3D surface topography used in diffuse tomographic reconstructions of internal sources.

Reflectant and transmissionfluorescence (reduction of autofluo-rescence).

Spectral unmixing algorithms allow detection and separation of multiple reporters, reduce the effects of tissue autofluorescence, reduce cross talk between reporters.

Transmission fluorescence imaging enables more sensitive detection and accurate quantification of deep sources, and to reduces the effects of autofluorescence.

IVIS-Spectrum

before

after 6 days

beforeafter

2 days 3 days 4 days 5 days 6 days

Narrow band excitation and emission filters and spectral unmixing algorithms allow to visualize multiple reporters in the same subject with reduced cross talk.

IVIS-Spectrum

IVIS-Lumina XRThe IVIS® Lumina XR can be used to image fluorescent and bioluminescent reporters both in vitro and in vivo, together with X-ray, giving the optical and X-ray overlap. Living Image software can be applied to obtain the data in anatomical context.

Mice injected with PC3M-luc cells (human prostatecancer). Metastasis were detected in the rib cage, lymph node (A), spinal cord and prostate(B), whichwas confirmed by necropsy analysis.

IVIS-kineticThe IVIS Kinetic provides a real time, fast imaging system enabling acquisition of biologically relevant events within milliseconds. It can perform both quantitativeluminescence and fluorescence.

7417.flv

Real-time luciferase activation in PC3M-luc tumors (human prostatecarcinoma) growing subcutaneously (image taken every 57 ms).

IVIS-kinetic

Real-time luciferase activation in PC3M-luc tumors (human prostatecarcinoma) investigated in walking mouse (image taken every 33 ms).

Skrót do 7419.lnk

Infection of common carp by a recombinantstrain of koi herpesvirus (KHV) expressing thefirefly luciferase.

To investigate the portal of entry of KHV, common carps were infected by bathing themin water containing the KHV LUC recombinant strain then were analyzed by IVIS at different time post-infection: examplesof day 3 and day 11 post-infection.

Berenice Costes, Victor Staline Raj, University of Liege

IVIS for fish

Application in oncology

Xenograft or allograft models of modified cell lines offer the ability to:• Quantify tumor burden in the whole mouse• Detect micrometastases with high sensitivity• Visualize metastases spontaneously generated from a primary tumor• Follow responses to therapeutic treatments non-invasively in longitudinal studies

Subcutaneous injection of PC-3M-luc-C6 cell line (prostate cancer model)

The subcutaneous tumor growth from an initial injection of “5” 4T1-luc2 cells in a nude mouse -monitoring of tumor development from implantation till the end of the study (tumors becomepulpable after ~28 days).

Application in oncologyHigh level of sensitivity allows to monitor early tumor behavior and detect micrometastases. Tumor growth may be measured before the tumors are palpable.

Detection of a single 4T1-luc2 cell (murine breast carcinoma) in a nude mouse using an IVIS Spectrum.

The B16-F10-luc-G5 mouse melanoma cells injected i.v., colonize the lung and form lesions that can be imaged non-invasively. Imaging results were compared to lesions counted on the surface of the lungs. A strong statistical association between mean bioluminescence and mean lung lesions was found (R2=0.9833).


Intravenous injection of murine melanoma cells: experimental metastatic model.


Metastasis formed by 4T1-luc2 cells (murine breastcarcinoma) 6 weeks afterinjection into fatpad.

Fatpad injection of murine breast cancer cell: experimental metastatic model.

The human VEGF promoter (2.3 kb) was fused to the Fluc gene and stably introduced into PC-3M cells (human prostate cancer). Subcutaneous tumors were established and imaging was used to monitor the expression of hVEGF-luc during the course of tumor growth as compared to SV-40-luc. In vivo imaging revealed activation of hVEGF1-luc relative to SV-40-luc during early stages of tumor development.

Application in oncologyMonitoring of angiogenic activity: induction of VEGF expression.

Heme-oxygenase-1 (HO-1) is an ubiquitous protein induced by toxins as well as oxidativestress or (in rodents) by hypoxia. It catalyzes the first and rate-limiting step in hemedegradation, acting as a cytoprotective, antiinflammatory and proangiogenic factor. A transgenic mice expressing luciferase under control of murine HO-1 promoter can beused to monitor toxicity due to a variety of agents.

Application in oncology: HO-1 promoter

Induction of luciferase reporter in mHo1-luc transgenic mouse following i.p. injection with doxorubicin.

P53 response elements were used to drive a minimal promoter fused to the firefly luciferase gene. A549 human tumor cell lines were stably transfected with this construct. When these cells were implanted subcutaneously into nude mice, the reporter was induced with the anti-cancer agent doxorubicin 6 h after challange.

Application in oncologyActivity of p53 transcription factor may be induced in response to chemotherapy andDNA damage.

Erika Spaeth, MD Anderson Cancer Center

Double labelling with Fluc and Rluc

MSC (labeled with adeno-viral expressing renillaluciferase) were injected 2 days prior to the imagingand can be seen localizingto the tumor.

4T1 (stably labeled withfirefly luciferase) tumor cells injected subcutane-ously into bilateral limbsof mice. Image is 3 weekspost injection-metastasesto the lungs can be seen inthe zoom-image.

Togel et al. Am J Physiol Renal Physiol 2008

Application in progenitor cell trafficking

MSC transfected with a luciferase reporter

healthy mousekidney injury

24 h after MSC injection

Yuan Cao, Stanford University

Hematopeosis from stem cells

Wang et al. Blood 2003

Expression of luciferase in hematopoietic stem cells:CD34+/CD38+ or CD34+/CD38-

Wang et al. Blood 2003

Longitunidal imaging of progenitor cells

CD34+/CD38+

CD34+/CD38-

CD34+/CD38-(scaled down)

Repopulation (3 weeks) is derived mainly from CD34+CD38+ cells whereas 90% of long termrepopulation is from the more rare and primitive CD34+CD38- cells.

Application in studies on inflammation: iNOS promoter

NO plays an important role in septic shock. Its vasodilatory effects are well known, but italso has pro- and anti-inflammatory properties, assumes crucial in antimicrobial hostdefense.

The iNos-luc transgenic mouse uses a 1.3 kb promoter fragment of the mouse iNOS gene to drive luciferase expression. Transgenic mice expressing luciferase under control of iNOS promoterwere injected with LPS, IFNγγγγ or both and then imaged 6 h after treatment.

Application in studies of inflammation

Mice were transiently transfectedwith a construct containing 7.9kb of the murine SAA promoter to drive luciferase expression. Eight days later they werechallenged with LPS.

SAA (Serum Amyloid A-1) is a liver specific acute phase response gene that is highly induced in both acute and chronic inflammatory diseases. In addition, it is an important component of the protein deposited in some amyloidosis disease states.

The NFκB is a ubiquitous transcription factor involved in infl ammation, and the major inducer of iNOS. The NFκB-luc transgenic mouse uses a set of response elements for NFκB to regulate a minimal promoter driving luciferase activity.

Application in studies on inflammation: NFκκκκB activity

NFκκκκB-luc transgenic mice were injectedwith LPS and then imaged after differenttime points.

CYP 3A4-luc transgenic mice: basal activity

The signal intensity is lower in females than in males (?!)

Cytochrome P450 enzymes are a superfamily containing proteins encoded by at least 57 P450 genes in humans.

P450 CYP 3A4 is responsible for oxidation of 50 to 60% of clinical drug sand is expressedprimarily in liver and small intestine CYP 3A4 induction b y xenobiotics occurs when a compoundbinds to the xenobiotic receptor PXR and the complex thendimerizes

Xhang et al., Drug Metabol Deposition 2003


There were clear gender differences, with signals greaterin male compared with female mice.

It is supposed that androgen response element (ARE)-likesequences existing in the 5'-flanking region of CYP 3A4 gene may be functionally activated by androgens in mice, but not in humans. This speculation was confirmed by implanting testosterone pellets subcutaneously in to femaleCYP 3A4-luc transgenic mice.


Application in studies on diabetes: insulin promoterMice or rats injection with streptozotocin (STZ) are one of the most frequently usedmodels of type-1 diabetes. STZ destroys b-cells in pancreas. Rip-luc transgenic mice contain luciferase gene under control of rat insulin promoter.

Male Rip-luc mice were injected i.v. with STZ or vehicle. The STZ-treated animals becomehyperglycemic within two days. Also the bioluminescent signal drops dramatically.

Wound healing model

Grochot-Przeczek et al. PLoS One 2009

WT

db/db

Application in studies on angiogenesis: VEGFR-2 promoter

Pounch wounds were generated in the skin of transgenic mice expressing luciferase undercontrol of VEGFR-2 promoter.

Angiogenesis is crucial for proper wound healing. The major proangiogenic factor isVEGF acting mostly through VEGFR2.

Application in infection diseases

Saeij et al. Infection and Immununity 2005

IVIS can be used for examination of in vivo growth, dissemination, and reactivation ofstrains of the protozoan parasiteToxoplasma gondii.

Two T. gondii strains, one with a highly virulent disease phenotype in mice (S23)and the otherwitha 1,000-fold-lower virulence phenotype (S22),were engineered to stably express theluciferase both in vitro and after injection in mice.

Comparison of progression of twoT. gondii strain tachyzoites, one with a highly virulent diseasephenotype in mice (S23)and the other witha 1,000-fold-lower virulence phenotype (S22).





control

Mice were infected with T. gondi, then treated with sulfalazine. Starting from day 80th someanimals were treated with immunosuppressant dexamethasone sulfate. This resulted in T. gondireactivation.

immunosuppression

Luciferase-engineered bacterial strains can be used for in vivo real-time monitoring of biofilms. An in vivo mouse model of device-related biofilm infection is established by subcutaneous implantation of either pre- or post-colonized catheters with biofilm-forming pathogens.

Monitoring of biofilm formation

Biofilm formed on catheters placed subcutaneously, colonized by bioluminescent strain ofStaphylococcus aureus.

Naik et al. PNAS 2007

The Wnt/ββββ-catenin (ββββ-cat) signal transduction pathway plays a key role in developmentand its activity is aberrant in different cancers.

In the absence of Wnt, ββββ-cat is phosphorylated, then ubiquitinated and degraded via theproteasome.

In the presence of Wnt,hypophosphorylated ββββ-cat is stabilized in the cytoplasm andtranslocates to the nucleus, where it forms a complex withT cell factor (TCF) orlymphoid enhancer-binding factor (LEF) to activate transcription of ~50 genes, including c-MYC or cyclin D1.

The ββββ-cat firefly luciferase (ββββ-cat-FLuc) or ββββ-cat click beetlegreen luciferase (ββββ-cat-promoter-CBG-luc) fusion reportersallow direct readout of ββββ-cat protein levels in real time. They can serve as tools for the characterization andpharmacodynamic analysis of potential regulators anddrugs that target the Wnt pathway via effects on ββββ-catstabilization.

Application in signal transduction study: ββββ-catenin

click beetle



rWnt3acontrol

Application in signal transduction studyProtein fragment complementation depends on division ofa monomeric enzyme intotwo separate components that do not spontaneously reassembleand function. Enzymeactivity occurs only upon complementation induced by the interaction of fused protein-binding partners. IVIS can be used for studies of such interactions in living animals.

Luker et al., PNAS 2004

Truncated N-terminal and C-terminalfragments of luciferase were fused to the rapamycin-binding domain (FRB) of the mTOR and FK506-binding protein (FKBP), respectively.

FRB-NLuc : CLuc-FKBP luciferasecomplementation imaging pairreconstituted luciferase activity incells upon single-site binding ofrapamycin.

Application in signal transduction study: mTOR

Luker et al., PNAS 2004

HEK 293 cells were cultured in a 96-well plate, transfected with proper plasmidsand stimulated with rapamycin for 6 h

HEK 293 cells transfected with proper plasmids, weremixed with matrigel and injected intrapritoneally to mice . Then mice were injected i.p. with rapamycin for 16 h.

ConclusionsIVIS can be used for studies of:

• Drug metabolism (monitoring the expression of key genes involved in drug metabolism)• Gene therapy vectors(biodistribution of potential vectors)• Formulations (Biodistribution of proprietary formulations and related drug delivery systems)• Stem cell research(tracking and monitoring stem cell populations)

Real-time in vivo imaging offers the advantage in:

• Oncology (monitoring tumor growth and metastasis, and the expression of key genes involved in oncology and related processes, such as angiogenesis or apoptosis)• Infectious diseases (tracking and monitoring bacterial, fungal, protozoal, and viral infections)• Immunology (monitoring the expression of key genes involved in inflammatory pathways and/or tracking and monitoring T- or B- lymphocytes).• Cardiology (monitoring the expression of genes involved in the cardiovascular and lipid metabolism pathways)• Diabetology (monitoring endogenous or transplanted pancreatic beta cells; monitoring the expression of key genes involved in the diabetes and obesity pathways)• Neurology (monitoring the expression of key genes involved in neurological processes such as neuroinflammation, neuroprogenitor cell lines and neurodegeneration)

Thank you

ivis - in vivo imaging system - jagiellonian...

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