In vivo Dual Substrate Bioluminescent Imaging
Case Comprehensive Cancer Center, Case Western Reserve University
Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.
Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay
1Department of Molecular and Cell Biology, Harvard, 2Molecular and Metabolic Signalling Centre, Division of Basic Medical Sciences, St. George's University of London
Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.
In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus
Experimental Pathology, University of Texas Medical Branch
Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.
A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo
1Department of Biomedical Engineering, University of Wisconsin-Madison, 2Department of Biomedical Engineering, Materials Science Program, Laboratory for Optical and Computational Instrumentation, University of Wisconsin-Madison
A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of ˜1,000 cells in peripheral tissues.
Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells
MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.
Induction and Testing of Hypoxia in Cell Culture
Center for Cell and Gene Therapy, Baylor College of Medicine
Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.
In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model
1Department of Radiation Oncology, University of Texas Southwestern Medical Center, 2Department of Radiology, University of Texas Southwestern Medical Center, 3Department of Radiation Oncology, Kyoto University Graduate School of Medicine
Bioluminescence imaging of hypoxia inducible factor-1α activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.
In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells
Departments of Radiology and Medicine (Cardiology), Stanford University School of Medicine
With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.
In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection
RNA Biology, New England Biolabs
This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.
Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging
Department of Neurological Surgery, University of California, San Francisco - UCSF
Luciferase-modified human brain tumor xenografts can be established intracranially in athymic mice, with subsequent monitoring of tumor growth and response to therapy using bioluminescence imaging. In combination with survival analysis, bioluminescence monitoring is an essential research tool for pre-clinical testing of therapies being considered for treating brain tumors.
An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells
Infectious Disease Research Center, CHUL (CHUQ), Quebec City, Quebec, Canada
We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.
Intracellular Refolding Assay
Institute of Toxicology and Genetics, Karlsruhe Institute of Technology
In this protocol a method to measure intracellular protein refolding after heat shock is described. This method can be used to study foldases like molecular chaperones and their co-factors or compounds able to influence their activity. Firefly luciferase activity is used as reporter to measure chaperone refolding activity.
In vivo Imaging of Transgenic Leishmania Parasites in a Live Host
1Interdisciplinary Immunology Program, University of Iowa, and the VA Medical Center, 2Department of Biochemistry, University of Iowa, and the VA Medical Center, 3Department of Internal Medicine, University of Iowa, 4Department of Molecular Microbiology, Washington University School of Medicine, 5Division of Dermatology, Harbor-UCLA Medical Center, Hanley-Hardison Research Center, 6Interdisciplinary Immunology Program, Iowa City VA Medical Center, 7Departments of Internal Medicine, Microbiology and Epidemiology, University of Iowa
An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.
In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum
Biology Research and Development , Caliper Life Sciences
Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.
Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging
Imaging Biology Research and Development, Caliper Life Sciences
An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.
Introduction to the Ultrasound Targeted Microbubble Destruction Technique
Department of Medicine, JABSOM, University of Hawaii
Ultrasound Targeted Microbubble Destruction (UTMD) can be used to direct site-specific delivery of bioactive molecules, including therapeutic genes, to target organs accessible to ultrasound, such as the heart and liver1-6.
Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection
Lindsley F. Kimball Research Institute, New York Blood Center
This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.
In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer
1Department of Medical Oncology, Women's Cancer Program, 2Transgenic Mouse Facility, Fox Chase Cancer Center
Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.
Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)
The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.
Purification of Hsp104, a Protein Disaggregase
Department of Biochemistry and Biophysics, University of Pennsylvania
Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.
Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems
1Tumour Virology Division F010, German Cancer Research Center (DKFZ), 2Inserm Unit 701, German Cancer Research Center (DKFZ)
Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).
Molecular Imaging to Target Transplanted Muscle Progenitor Cells
1Imaging Program, Lawson Health Research Institute, 2Department of Anatomy and Cell Biology, Western University, 3Department of Medical Biophysics, Western University
A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.
Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model
Department of Surgery, The Ohio State University Medical Center
Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.
Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents
1Department of Pathology, University of Alabama at Birmingham - UAB, 2Department of Radiology, University of Alabama at Birmingham - UAB, 3Department of Cell Biology and Neurobiology, University of Alabama at Birmingham - UAB
A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.
Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors
1Division of Experimental Virology, Twincore, Centre for Experimental and Clinical Infection Research, 2Aaron Diamond AIDS Research Center, Laboratory of Retrovirology, The Rockefeller University, NY
We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.
Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus
Swedish Medical Nanoscience Center, Department of Neuroscience, Karolinska Institutet
The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.
Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and and Induced Pluripotent Stem Cells (iPSCs)
1Department of Medicine (Hematology, Oncology, and Transplant), University of Minnesota, Minneapolis, 2Stem Cell Institute, University of Minnesota, Minneapolis
This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.
Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice
1Laboratory of Experimental Hematology, University of Antwerp, 2Bio Imaging Lab, University of Antwerp
This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.
Ex Vivo Infection of Live Tissue with Oncolytic Viruses
Center for Innovative Cancer Research, Ottawa Hospital Research Institute (OHRI)
Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.
Using Luciferase to Image Bacterial Infections in Mice
Microbial & Molecular Pathogenesis, Texas A&M Health Science Center
Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.
Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding
Department of Pharmacology, School of Medicine, New York University
The crown region of different V3 loop sequences of the surface envelope glycoprotein (gp120) of HIV-1 can be structurally characterized in many cases by in silico folding of positions 10 to 22 of the loop using a state-of-the-art ab initio folding algorithm. Here we demonstrate the folding and evaluation of this region of the V3 loop from the R2 strain of HIV-1, a uniquely neutralization sensitive strain with puzzling functional properties.
Models of Bone Metastasis
1Department of Pharmacology, Vanderbilt University, 2Vanderbilt Center for Bone Biology, Vanderbilt University, 3Department of Veterans Affairs, Tennessee Valley Healthcare System (VISN 9), 4Department of Medicine, Division of Clinical Pharmacology, Vanderbilt University, 5Department of Cancer Biology, Vanderbilt University
Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.
Quantitative Measurement of the Immune Response and Sleep in Drosophila
Center for Sleep and Circadian Neurobiology, University of Pennsylvania Perelman School of Medicine
To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NFκB.
Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)
1Transplant and Stem Cell Immunobiology Lab (TSI) and CVRC, University Hospital Hamburg, University Heart Center Hamburg, 2Department of Experimental and Clinical Pharmacology and Toxicology, University Heart Center Hamburg, 3CT Surgery, Stanford University School of Medicine
This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.
Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters
Department of Biological Sciences, The University of Memphis
Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.
RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells
Department of Microbiology, Penn Genome Frontiers Institute, University of Pennsylvania
Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.
An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment
1Department of Urology and Helen Diller Comprehensive Cancer Center, University of California, San Francisco, 2Alnylam Pharmaceuticals, Inc.
Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.
Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice
The Walter and Eliza Hall Institute of Medical Research
A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.
DNA Vector-based RNA Interference to Study Gene Function in Cancer
1Department of Cancer Biology and Comprehensive Cancer Center, Wake Forest University School of Medicine, 2Department of Pathology and Comprehensive Cancer Center, Wake Forest University School of Medicine
RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo.
A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model
1Molecular Virology and Gene Therapy, KU Leuven, 2Department of Woman and Child, KU Leuven, 3Neurobiology and Gene Therapy, KU Leuven, 4Division of Nuclear Medicine, KU Leuven, 5Biomedical NMR Unit/ MoSAIC, KU Leuven
We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.
Alphavirus Transducing System: Tools for Visualizing Infection in Mosquito Vectors
Microbiology, Immunology, and Pathology, Colorado State University
Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.
Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-κB Reporter Transgenic
1Cancer Biology Department, Vanderbilt University Medical Center, 2Department of Medicine, Vanderbilt University Medical Center, 3Department of Pharmaceutical Sciences, University of Hawaii at Hilo College of Pharmacy
Described here is a technique in which lipopolysaccharide is injected into the lactating mouse mammary gland via the nipple to simulate mastitis, a condition commonly caused by bacterial infection. Lipopolysaccharide injection results in increased nuclear factor kappa B (NF-κB) signaling, visualized through bioluminescent imaging of an NF-κB luciferase reporter mouse.
MISSION esiRNA for RNAi Screening in Mammalian Cells
Max Planck Institute of Molecular Cell Biology and Genetics
Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.
Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia
1Division of Cardiovascular Medicine, Stanford University, 2Department of Radiology, Stanford University
The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.
Genomic Transformation of the Picoeukaryote Ostreococcus tauri
1SynthSys, University of Edinburgh, 2Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, Paris 06, 3UMR 7621, Laboratoire d'Océanographie Microbienne, Observatoire Océanologique, Banyuls-sur-Mer, Université Pierre et Marie Curie, Paris 06
This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.
Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice
1Department of Microbiology and Immunology, University of Melbourne, 2Laboratory of Pediatric Infectious Diseases, Radboud University Nijmegen Medical Centre, 3The Centre for Dynamic Imaging, The Walter and Eliza Hall Institute for Medical Research
A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.
Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences
We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.
Murine Bioluminescent Hepatic Tumour Model
1Cork Cancer Research Centre, Mercy University Hospital and Leslie C. Quick Jnr. Laboratory, University College Cork, 2Department of Computer Science, University College Cork, 3South Infirmary Victoria University Hospital
This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.
Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure
1Department of Radiology, Massachusetts General Hospital, Harvard Medical School, 2Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Harvard Medical School, 3Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, 4Department of Medicine, Massachusetts General Hospital, Harvard Medical School, 5Center for biotechnology and Informatics, The Methodist Hospital Research Institute, 6Department of Radiology, The Methodist Hospital, Weill Cornell Medical College, 7Bejing University of Chinese Medicine, 8Department of Health Technology and Informatics, The Hong Kong Polytechnic University, 9Department of Radiology, Brigham and Women's Hospital, Harvard Medical School
Gua Sha, traditional Chinese therapeutic skin scraping, causes subcutaneous microvascular blood extravasation. We report a protocol of bioluminescence imaging of HO-1-luciferase transgenic mice to demonstrate that Gua Sha upregulates heme oxygenase-1 (HO-1) in multiple organs.
Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas
1Neuro-Oncology Research, Barrow Neurological Institute of St. Joseph’s Hospital and Medical Center, 2Neurosurgery Research Laboratory, Barrow Neurological Institute of St. Joseph’s Hospital and Medical Center
Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.
