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University of California, Irvine

61 ARTICLES PUBLISHED IN JoVE

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Biology

Injection of dsRNA into Female A. aegypti Mosquitos
Brian M. Luna 1, Jennifer Juhn 1, Anthony A. James 2
1Department of Molecular Biology and Biochemistry, University of California, Irvine (UCI), 2Department of Molecular Biology and Biochemistry, Department of Microbiology and Molecular Genetics, University of California, Irvine (UCI)

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus. The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated.

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Biology

Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes
Olle Terenius 1, Jennifer Juhn 1, Anthony A. James 2
1Department of Molecular Biology and Biochemistry, University of California, Irvine (UCI), 2Department of Molecular Biology and Biochemistry, Department of Microbiology and Molecular Genetics, University of California, Irvine (UCI)

Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.

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Biology

Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes
Nijole Jasinskiene 1, Jennifer Juhn 1, Anthony A. James 2
1Department of Molecular Biology and Biochemistry, University of California, Irvine (UCI), 2Department of Molecular Biology and Biochemistry, Department of Microbiology and Molecular Genetics, University of California, Irvine (UCI)

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

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Biology

Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes
Judy Coleman 1, Jennifer Juhn 1, Anthony A. James 2
1Department of Molecular Biology and Biochemistry, University of California, Irvine (UCI), 2Department of Molecular Biology and Biochemistry, Department of Microbiology and Molecular Genetics, University of California, Irvine (UCI)

The mosquito midgut and salivary glands are key entry and exit points for vector pathogens like Plasmodium falciparum and the dengue virus. This video demonstrates the dissection techniques for removing the midgut and salivary glands from Aedes aegypti mosquitoes.

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Biology

Preventing the Spread of Malaria and Dengue Fever Using Genetically Modified Mosquitoes
Anthony A. James 1
1Department of Molecular Biology and Biochemistry, Department of Microbiology and Molecular Genetics, University of California, Irvine (UCI)

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission. Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

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Biology

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit
Shenyuan Zhang 1, Michael D. Cahalan 1
1Department of Physiology and Biophysics, University of California, Irvine (UCI)

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

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Biology

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method
Alexandrine Froger 1, James E. Hall 1
1Department of Physiology and Biophysics, University of California, Irvine (UCI)

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

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Biology

Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice
Melanie P. Matheu 1, Ian Parker 2, Michael D. Cahalan 1
1Department of Physiology and Biophysics, University of California, Irvine (UCI), 2Department of Neurobiology and Behaviour, University of California, Irvine (UCI)

Two-photon imaging has uncovered lymphocyte motility and cellular interactions within the lymph node under basal conditions and durring an immune response 1. Here, we demonstrate adoptive transfer of T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node.

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Biology

Windowing Chicken Eggs for Developmental Studies
Matthew J. Korn 1, Karina S. Cramer 1
1Department of Neurobiology and Behaviour, University of California, Irvine (UCI)

The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling. In this article we demonstrate one egg preparation method that has been optimized for long survival times.

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Biology

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos
Matthew J. Korn 1, Karina S. Cramer 1
1Department of Neurobiology and Behaviour, University of California, Irvine (UCI)

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

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Biology

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification
Melanie P. Matheu 1, Michael D. Cahalan 1
1Department of Physiology and Biophysics, University of California, Irvine (UCI)

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically ≥ 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

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Biology

Murine Skin Transplantation
Kym R. Garrod 1, Michael D. Cahalan 1
1Department of Physiology and Biophysics, University of California, Irvine (UCI)

Allogeneic skin transplantation is a standard model to assay host T cell responses to MHC-disparate donor antigens. This video-article provides a visual tutorial of each step involved in performing a BALB/c-->C57BL/6 skin transplant.

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Biology

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells
Kitchener Wilson 1, Jin Yu 1, Andrew Lee 1, Joseph C. Wu 1
1Departments 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.

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Biology

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging
Melanie P. Matheu 1, Debasish Sen 1, Michael D Cahalan 1, Ian Parker 2
1Department of Physiology and Biophysics, University of California, Irvine (UCI), 2Department of Neurobiology and Behaviour, University of California, Irvine (UCI)

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

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Biology

Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH
Melanie P. Matheu 1, Christine Beeton 1, Ian Parker 2, K. George Chandy 1, Michael D. Cahalan 1
1Department of Physiology and Biophysics, University of California, Irvine (UCI), 2Department of Neurobiology and Behavior, University of California, Irvine (UCI)

Here we demonstrate a method for inducing and recording the progress of a delayed type-hypersensitivity (DTH) reaction in the rat ear. This is followed by a demonstration of the preparation of rat ear tissue for two-photon imaging of the effector / memory T cell response.

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Neuroscience

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures
Ardeshir S. Rahman 1, Shaudee Parvinjah 1, Michael A. Hanna 1, Pablo R. Helguera 1, Jorge Busciglio 1
1Department of Neurobiology and Behavior, University of California, Irvine

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

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Neuroscience

Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus
Kevin S. Carbajal 1,2, Jason G. Weinger 1,2, Lucia M. Whitman 1,2, Chris S. Schaumburg 1,2, Thomas E. Lane 1,2,3
1Department of Molecular Biology and Biochemistry, University of California, Irvine, 2Sue and Bill Gross Stem Cell Center, University of California, Irvine, 3Institute for Immunology, University of California, Irvine

The transplantation of mouse neural stem cells (NSCs) into the spinal cords of mice with established demyelination is detailed. The preparation of NSCs, the laminectomy of thoracic vertebra 9 (T9), and transplantation of NSCs is outlined along with the pre- and post-operative care of the mice.

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Neuroscience

Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation
Taruna Ikrar 1, Nicholas D. Olivas 1, Yulin Shi 1, Xiangmin Xu 1,2
1Department of Anatomy and Neurobiology, School of Medicine, University of California, Irvine, 2Department of Biomedical Engineering, School of Engineering, University of California, Irvine

This paper introduces an approach of combining laser scanning photostimulation with whole cell recordings in transgenic mice expressing GFP in limited inhibitory neuron populations. The technique allows for extensive mapping and quantitative analysis of local synaptic circuits of specific inhibitory cortical neurons.

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Biology

Quantifying Agonist Activity at G Protein-coupled Receptors
Frederick J. Ehlert 1, Hinako Suga 2, Michael T. Griffin 3
1Department of Pharmacology, University of California, Irvine, 2Department of Pharmacology, University of California, 3Schmid College of Science, Chapman University

A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation.

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Biology

Isolation and Purification of Kinesin from Drosophila Embryos
Robilyn Sigua 1, Suvranta Tripathy 1, Preetha Anand 1, Steven P. Gross 1
1Department of Developmental and Cell Biology, School of Biosciences, University of California, Irvine

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

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Immunology and Infection

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes
Jennifer Juhn 1, Anthony A. James 1,2
1Department of Molecular Biology and Biochemistry, University of California, Irvine, 2Department of Microbiology and Molecular Genetics, University of California, Irvine

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

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Medicine

A Contusion Model of Severe Spinal Cord Injury in Rats
Vibhor Krishna 1, Hampton Andrews 1, Xing Jin 2, Jin Yu 1, Abhay Varma 1, Xuejun Wen 3, Mark Kindy 1
1Department of Neuroscience, Division of Neurosurgery, Medical University of South Carolina, 2Bioengineering, Clemson University, 3Clemson-MUSC Bioengineering Joint Program

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

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Neuroscience

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve
Michelle R. Allen-Sharpley 1, Michelle Tjia 1, Karina S. Cramer 1
1Department of Neurobiology and Behavior, University of California, Irvine

Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain.

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Behavior

Creating Dynamic Images of Short-lived Dopamine Fluctuations with lp-ntPET: Dopamine Movies of Cigarette Smoking
Evan D. Morris 1,2,3,4, Su Jin Kim 1,3, Jenna M. Sullivan 1,3,4, Shuo Wang 3,4, Marc D. Normandin 5, Cristian C. Constantinescu 6, Kelly P. Cosgrove 1,2,3
1Diagnostic Radiology, Yale University, 2Psychiatry, Yale University, 3Yale PET Center, Yale University, 4Biomedical Engineering, Yale University, 5Nuclear Medicine, Massachusetts General Hospital, 6Radiological Sciences, University of California, Irvine

We present a novel PET imaging approach for capturing dopamine fluctuations induced by cigarette smoking. Subjects smoke in the PET scanner. Dynamic PET images are modeled voxel-by-voxel in time by lp-ntPET, which includes a time-varying dopamine term. The results are 'movies' of dopamine fluctuations in the striatum during smoking.

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Medicine

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection
Melissa F. Davis *1,2, Christopher Lay *1,2,3, Ron D. Frostig 1,2,3,4
1Department of Neurobiology & Behavior, University of California, Irvine, 2The Center for the Neurobiology of Learning and Memory, University of California, Irvine, 3The Center for Hearing Research, University of California, Irvine, 4Department of Biomedical Engineering, University of California, Irvine

We describe a highly reproducible method for the permanent occlusion of a rodent major cerebral blood vessel. This technique can be accomplished with very little peripheral damage, minimal blood loss, a high rate of long-term survival, and consistent infarct volume commensurate with the human clinical population.

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Engineering

Process of Making Three-dimensional Microstructures using Vaporization of a Sacrificial Component
Du T. Nguyen 1, Y. T. Leho 2, Aaron P. Esser-Kahn 2
1Department of Physics, University of California, Irvine, 2Department of Chemistry, University of California, Irvine

The Vaporization of a Sacrificial Component (VaSC) process is used to fabricate microvascular structures. This procedure uses sacrificial poly(lactic) acid fibers to form hollow microchannels with precise 3D geometric positioning provided by laser micromachined guide plates.

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Biology

Assessing Differences in Sperm Competitive Ability in Drosophila
Shu-Dan Yeh 1, Carolus Chan 1, José M. Ranz 1
1Department of Ecology and Evolutionary Biology, University of California, Irvine

Differential sperm competitive ability among Drosophila males with distinct genotypes can be ascertained through double-mating experiments. Each of these experiments involves one of the males of interest and a reference male. Readily identifiable markers in the progeny allow inference of the fraction of individuals fathered by each male.

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Engineering

Laser-induced Breakdown Spectroscopy: A New Approach for Nanoparticle's Mapping and Quantification in Organ Tissue
Lucie Sancey 1, Vincent Motto-Ros 2, Shady Kotb 1, Xiaochun Wang 3, François Lux 1, Gérard Panczer 3, Jin Yu 2, Olivier Tillement 1
1ILM-FENNEC UMR 5306, CNRS - Université Lyon 1, 2ILM-PUBLI UMR 5306, CNRS - Université Lyon 1, 3ILM-SOPRANO UMR 5306, CNRS - Université Lyon 1

Laser-induced breakdown spectroscopy performed on thin organ and tumor tissue successfully detected natural elements and artificially injected gadolinium (Gd), issued from Gd-based nanoparticles. Images of chemical elements reached a resolution of 100 μm and quantitative sub-mM sensitivity. The compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of a same biological tissue.

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Immunology and Infection

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages
Jing Wu 1, Roberta Pugh 1, Richard C. Laughlin 1, Helene Andrews-Polymenis 2, Michael McClelland 3, Andreas J. Bäumler 4, L. Garry Adams 1
1Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, 2Department of Microbial and Molecular Pathogenesis, College of Medicine, Texas A&M University System Health Science Center, 3University of California, Irvine, 4Department of Medical Microbiology & Immunology, School of Medicine, University of California, Davis

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

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Bioengineering

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles
Andrea Anzalone *1, Paolo Annibale *1, Enrico Gratton 1
1Biomedical Engineering, Laboratory for Fluorescence Dynamics, University of California, Irvine

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

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Biology

Generation of Transgenic Hydra by Embryo Microinjection
Celina E. Juliano 1, Haifan Lin 1, Robert E. Steele 2
1Yale Stem Cell Center and Department of Cell Biology, Yale University School of Medicine, 2Department of Biological Chemistry and the Developmental Biology Center, University of California, Irvine

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.

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Bioengineering

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope
Carmine Di Rienzo 1,2, Enrico Gratton 3, Fabio Beltram 1, Francesco Cardarelli 2
1NEST Laboratory, Scuola Normale Superiore, 2Center for Nanotechnology Innovation, Instituto Italiano di Tecnologia, 3Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

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Biology

Imaging Local Ca2+ Signals in Cultured Mammalian Cells
Jeffrey T. Lock 1, Kyle L. Ellefsen 1, Bret Settle 1, Ian Parker 1,2, Ian F. Smith 1
1Neurobiology and Behavior, University of California, Irvine, 2Physiology and Biophysics, University of California, Irvine

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

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Neuroscience

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord
Jason G. Weinger 1, Milton L. Greenberg 2, Melanie P. Matheu 4, Ian Parker 3, Craig M. Walsh 1, Thomas E. Lane 5, Michael D. Cahalan 2
1Molecular Biology and Biochemistry, University of California, Irvine, 2Physiology and Biophysics, University of California, Irvine, 3Neurobiology and Behavior, University of California, Irvine, 4University of California San Francisco Diabetes Center, University of California, San Francisco, 5Pathology, University of Utah

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

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JoVE Core

SSVEP-based Experimental Procedure for Brain-Robot Interaction with Humanoid Robots
Jing Zhao 1, Wei Li 1,2, Xiaoqian Mao 1, Mengfan Li 1
1School of Electrical Engineering and Automation, Tianjin University, 2Department of Computer & Electrical Engineering and Computer Science, California State University

The overall goal of this method is to establish an SSVEP-based experimental procedure by integrating multiple software programs to enable the study of brain-robot interaction with humanoid robots, which is prospective in assisting the sick and elderly as well as performing unsanitary or dangerous jobs.

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Neuroscience

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings
Kyle J. McCulloch 1, Daniel Osorio 2, Adriana D. Briscoe 1
1Department of Ecology and Evolutionary Biology, University of California, Irvine, 2School of Life Sciences, University of Sussex

The electrophysiological technique of intracellular recording is demonstrated and used to determine spectral sensitivities of single photoreceptor cells in the compound eye of a butterfly.

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Cancer Research

Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm
Thanh Kim Nguyen 1, Sarah J. Morse 1, Angela G. Fleischman 1
1Department of Medicine, Division of Hematology/Oncology, University of California, Irvine

This manuscript provides a description of the methodology used to establish transduction-transplantation mouse models. A detailed account is given of technical errors to avoid when performing bone marrow transplants. A clear understanding should be gained of the importance of high viral titer, transfection/transduction, and irradiation.

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Genetics

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus
Ira L. Blitz 1
1Department of Developmental and Cell Biology, University of California, Irvine

Genes essential for survival pose technical hurdles for creating mutant lines. Leapfrogging circumvents lethality by combining genome editing with primordial germ cell transplantation to create wild-type animals carrying germline mutations. Leapfrogging also permits the efficient generation of homozygous null mutants in the F1 generation. Here, the transplantation step is demonstrated.

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Genetics

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage
Xiangduo Kong *1, Gladys M.S. Cruz *2, Bárbara A. Silva 2, Nicole M. Wakida 2, Nima Khatibzadeh 2, Michael W. Berns 2,3,4, Kyoko Yokomori 1
1Department of Biological Chemistry, School of Medicine, University of California, Irvine, 2Beckman Laser Institute and Medical Clinic, University of California, Irvine, 3Department of Developmental and Cell Biology, School of Biological Sciences, University of California, Irvine, 4Department of Biomedical Engineering and Surgery, University of California, Irvine

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

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Bioengineering

A Rapid Image-based Bacterial Virulence Assay Using Amoeba
Kumar Perinbam 1, Albert Siryaporn 1,2
1Department of Physics and Astronomy, University of California, 2Department of Molecular Biology and Biochemistry, University of California

Here, we present a protocol to measure the virulence of planktonic or surface-attached bacteria using D. discoideum (amoeba) as a host. Virulence is measured over a period of 1 h and host killing is quantified using fluorescence microscopy and image analysis. We demonstrate this protocol using the bacterium P. aeruginosa.

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Biology

Assessment of Zebrafish Lens Nucleus Localization and Sutural Integrity
Irene Vorontsova 1,2, James E. Hall 1, Thomas F. Schilling 2
1Department of Physiology and Biophysics, University of California, Irvine, 2Department of Developmental and Cell Biology, University of California, Irvine

These protocols were developed to analyze cortical lens morphology, structural integrity of the zebrafish lens sutures in fixed and live lenses and to measure the position of the zebrafish lens nucleus along the anterior-posterior axis.

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Bioengineering

Databases to Efficiently Manage Medium Sized, Low Velocity, Multidimensional Data in Tissue Engineering
Alexander R. Ochs 1,2, Mehrsa Mehrabi 1,2, Danielle Becker 1,2, Mira N. Asad 1,2, Jing Zhao 1,2, Michael V. Zaragoza 3,4, Anna Grosberg 1,2,5,6,7
1Department of Biomedical Engineering, University of California, Irvine, 2The Edwards Lifesciences Center for Advanced Cardiovascular Technology, University of California, Irvine, 3Pediatrics-Genetics & Genomics Division-School of Medicine, University of California, Irvine, 4Biological Chemistry-School of Medicine, University of California, Irvine, 5Department of Chemical and Biomolecular Engineering, University of California, Irvine, 6Center for Complex Biological Systems, University of California, Irvine, 7The NSF-Simons Center for Multiscale Cell Fate Research (CMCF), University of California, Irvine

Many researchers generate "medium-sized", low-velocity, and multi-dimensional data, which can be managed more efficiently with databases rather than spreadsheets. Here we provide a conceptual overview of databases including visualizing multi-dimensional data, linking tables in relational database structures, mapping semi-automated data pipelines, and using the database to elucidate data meaning.

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Neuroscience

Microinjectrode System for Combined Drug Infusion and Electrophysiology
M. Isabel Vanegas 1, Kenneth R. Hubbard 1,2, Rahim Esfandyarpour 3,4, Behrad Noudoost 1
1Department of Ophthalmology and Visual Sciences, University of Utah, 2Department of Biomedical Engineering, University of Utah, 3Department of Electrical Engineering and Computer Science, University of California, Irvine, 4Department of Biomedical Engineering, University of California, Irvine

We present a microinjectrode system designed for electrophysiology and assisted delivery of experimental probes (i.e., nanosensors, microelectrodes), with optional drug infusion. Widely available microfluidic components are coupled to a cannula containing the probe. A step-by-step protocol for microinjectrode construction is included, with results during muscimol infusion in macaque cortex.

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Environment

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae
Bin Zhang 1,2, Xinyue Yang 2, Jing Zhao 3, Ting Xu 2,4, Daqiang Yin 2,4
1State Key Laboratory of Marine Geology, Tongji University, 2Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Engineering, Tongji University, 3Shanghai Collaborative Innovation Centre for WEEE Recycling, WEEE Research Center of 10 Shanghai Polytechnic University, 4Shanghai Institute of Pollution Control and Ecological Security

A detailed experimental protocol is presented in this paper for the evaluation of neurobehavioral toxicity of environmental pollutants using a zebrafish larvae model, including the exposure process and tests for neurobehavioral indicators.

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Biology

Time-lapse Imaging of Bacterial Swarms and the Collective Stress Response
Jean-Louis Bru 1, Albert Siryaporn 1,2, Nina Molin Høyland-Kroghsbo 3
1Department of Molecular Biology & Biochemistry, University of California, Irvine, 2Department of Physics & Astronomy, University of California, Irvine, 3Department of Veterinary and Animal Sciences, University of Copenhagen

We detail a simple method to produce high-resolution time-lapse movies of Pseudomonas aeruginosa swarms that respond to bacteriophage (phage) and antibiotic stress using a flatbed document scanner. This procedure is a fast and simple method for monitoring swarming dynamics and may be adapted to study the motility and growth of other bacterial species.

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JoVE Journal

A Bioinformatics Pipeline for Investigating Molecular Evolution and Gene Expression using RNA-seq
Aide Macias-Muñoz 1, Ali Mortazavi 1
1Department of Developmental and Cell Biology, University of California, Irvine

The purpose of this protocol is to investigate the evolution and expression of candidate genes using RNA sequencing data.

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Bioengineering

Building a Simple and Versatile Illumination System for Optogenetic Experiments
Phillip Kyriakakis 1, Lourdes Fernandez de Cossio 2, Patrick Wade Howard 1, Sivleng Kouv 1, Marianne Catanho 1, Vincent J. Hu 3, Robert Kyriakakis 1, Molly E. Allen 1, Yunhan Ma 4, Marcelo Aguilar-Rivera 1, Todd P. Coleman 1
1Department of Bioengineering, University of California, San Diego, 2University of California, San Diego, 3Department of Mathematical Computational, and Systems biology, University of California, Irvine, 4Department of Biomedical Engineering, Duke University

This protocol describes how to perform optogenetic experiments for controlling gene expression with red and far-red light using PhyB and PIF3. Included are step-by-step instructions for building a simple and flexible illumination system, which enables the control of gene expression or other optogenetics with a computer.

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Genetics

Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae
Beth Crawford Poulton 1, Fraser Colman 1, Amalia Anthousi 1,2,3, Linda Grigoraki 1, Adriana Adolfi 1, Amy Lynd 1, Gareth John Lycett 1
1Department of Vector Biology, Liverpool School of Tropical Medicine, 2IMBB FORTH, 3Department of Biology, University of Crete

The bipartite GAL4-UAS system is a versatile tool for modification of gene expression in a controlled spatiotemporal manner which permits functional genetic analysis in Anopheles gambiae. The procedures described for using this system are a semi-standardized cloning strategy, sexing and screening of pupae for fluorescent protein markers and embryo fixation.

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Genetics

Site-Directed φC31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria
Adriana Adolfi 1,2, Amy Lynd 1, Gareth J. Lycett 1, Anthony A. James 2,3
1Vector Biology Department, Liverpool School of Tropical Medicine, 2Department of Microbiology & Molecular Genetics, University of California, 3Department of Molecular Biology & Biochemistry, University of California

The protocol describes how to achieve site-directed modifications in the genome of Anopheles malaria mosquitoes using the φC31 system. Modifications described include both the integration and the exchange of transgenic cassettes in the genome of attP-bearing docking lines.

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Biochemistry

Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction
Joann Phan 1, Joseph Kapcia III 1, Cynthia I. Rodriguez 2, Victoria L. Vogel 3, Daniel B Cardin 3, Sage J. B. Dunham 3, Katrine Whiteson 1
1Department of Molecular Biology and Biochemistry, University of California Irvine, 2Department of Ecology and Evolutionary Biology, University of California Irvine, 3Entech Instruments Inc.

This protocol describes the extraction of volatile organic compounds from a biological sample with the vacuum-assisted sorbent extraction method, gas chromatography coupled with mass spectrometry using the Entech Sample Preparation Rail, and data analysis. It also describes culture of biological samples and stable isotope probing.

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Genetics

Small-Cage Laboratory Trials of Genetically-Engineered Anopheline Mosquitoes
Rebeca Carballar-Lejarazú 1, Thai Binh Pham 2, Vanessa Bottino-Rojas 1, Adriana Adolfi 1,3, Anthony A. James 1,2
1Department of Microbiology & Molecular Genetics, University of California, Irvine, 2Department of Molecular Biology & Biochemistry, University of California, Irvine, 3Vector Biology Department, Liverpool School of Tropical Medicine

The protocols reported here illustrate three alternative ways to assess the performance of genetically-engineered mosquitoes destined for vector control in laboratory-contained small cage trials. Each protocol is tailored to the specific modification the mosquito strain bears (gene drive or non-gene drive) and the types of parameters measured.

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Genetics

Microinjection Method for Anopheles gambiae Embryos
Rebeca Carballar-Lejarazú 1, Taylor Tushar 1, Thai Binh Pham 2, Anthony A. James 1,2
1Department of Microbiology & Molecular Genetics, University of California, Irvine, 2Department of Molecular Biology & Biochemistry, University of California, Irvine

Microinjection techniques are essential to introduce exogenous genes into the genomes of mosquitoes. This protocol explains a method used by the James laboratory to microinject DNA constructs into Anopheles gambiae embryos to generate transformed mosquitoes.

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Genetics

Digital-Droplet PCR to Detect Indels Mutations in Genetically Modified Anopheline Mosquito Populations
Rebeca Carballar-Lejarazú 1, Thai Binh Pham 2, Adam Kelsey 1, Taylor Tushar 1, Anthony A. James 1,2
1Department of Microbiology & Molecular Genetics, University of California, Irvine, 2Department of Molecular Biology & Biochemistry, University of California, Irvine

This protocol provides the steps from DNA extraction to experimental set-up for digital droplet PCR (ddPCR), including analysis for the identification and quantification of non-homologous end-joining (NHEJ) events at target sites following gRNA-induced Cas9 cleavage and DNA repair. Other uses of this method include applications such as polymorphism detection and gene-editing variant verification.

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Biology

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection
Jing Zhao 1,2,3, Jie Yang 1,2,3, Lili Zhang 1,2, Rongxiang Fang 1,2, Yan Huo 1,2
1State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, 2National Plant Gene Research Center, 3University of the Chinese Academy of Sciences

The present protocol describes a method to collect sufficient saliva from piercing-sucking insects using an artificial medium. This is a convenient method for collecting insect saliva and studying salivary function on insect feeding behavior and vector-borne virus transmission.

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Medicine

Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome
Jacob Vieira 1, Tara Gallagher 2, Hui-Yu Sui 1, Sirus Jesudasen 3, Katrine Whiteson 2, George A. O'Toole 4, Kurt Hanselmann 5, Peggy S. Lai 1
1Division of Pulmonary and Critical Care Medicine, Massachusetts General Hospital, 2Department of Molecular Biology & Biochemistry, University of California, Irvine, 3Department of Medicine, Massachusetts General Hospital, 4Department of Microbiology & Immunology, Geisel School of Medicine at Dartmouth, 5Swiss i-research and teaching institute

The goal of this protocol is to develop a model system for the effect of hyperoxia on cystic fibrosis airway microbial communities. Artificial sputum medium emulates the composition of sputum, and hyperoxic culture conditions model the effects of supplemental oxygen on lung microbial communities.

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Biology

Structure-Based Simulation and Sampling of Transcription Factor Protein Movements along DNA from Atomic-Scale Stepping to Coarse-Grained Diffusion
Chao E *1, Liqiang Dai *1,2, Jiaqi Tian 3,4, Lin-Tai Da 4, Jin Yu 5,6,7
1Beijing Computational Science Research Center, 2Shenzhen JL Computational Science and Applied Research Institute, 3School of Medical Informatics and Engineering, Xuzhou Medical University, 4Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, 5Department of Physics and Astronomy, University of California, Irvine, 6Department of Chemistry, University of California, Irvine, 7NSF-Simons Center for Multiscale Cell Fate Research, University of California, Irvine

The goal of this protocol is to reveal structural dynamics of one-dimensional diffusion of protein along DNA, using a plant transcription factor WRKY domain protein as an exemplary system. To do this, both atomistic and coarse-grained molecular dynamics simulations along with extensive computational samplings have been implemented.

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Neuroscience

Full- versus Sub-Regional Quantification of Amyloid-Beta Load on Mouse Brain Sections
Yuu Ohno 1, Riley Murphy 2, Matthew Choi 3, Weijun Ou 4, Rachita K. Sumbria 4,5
1Henry E. Riggs School of Applied Life Sciences, Keck Graduate Institute, 2Crean College of Health and Behavioral Sciences, Chapman University, 3Keck Science Department, Claremont McKenna College, 4Department of Biomedical and Pharmaceutical Sciences, School of Pharmacy, Chapman University, 5Department of Neurology, University of California, Irvine

The present protocol describes and compares the procedure to perform a full-region or sub-region of interest analysis of sagittal mouse brain sections to quantify amyloid-beta load in the APP/PS1 transgenic mouse model of Alzheimer's disease.

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Neuroscience

Head Implants for the Neuroimaging of Awake, Head-Fixed Rats
Mehwish Bhatti 1, Hayden Malone 1, Gabriel Hui 1, Ron D. Frostig 1,2,3
1Department of Neurobiology and Behavior, School of Biological Sciences, University of California Irvine, 2Department of Biomedical Engineering, School of Engineering, University of California, Irvine, 33Center for Neurobiology of Learning and Memory, University of California, Irvine

A detailed new procedure for functional imaging of awake, head-fixed rats is described.

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Bioengineering

Light-Induced Dielectrophoresis for Characterizing the Electrical Behavior of Human Mesenchymal Stem Cells
Kiara L. Lacy 1, Samuel Salib 1, Mary Tran 2, Tunglin Tsai 1, Rominna Valentine 1, Herdeline Ann M. Ardoña 1,2,3,4, Tayloria N. G. Adams 1,2,4
1Department of Chemical and Biomolecular Engineering, Samueli School of Engineering, University of California, Irvine, 2Department of Biomedical Engineering, Samueli School of Engineering, University of California, Irvine, 3Department of Chemistry, School of Physical Sciences, University of California, Irvine, 4Sue & Bill Gross Stem Cell Research Center, University of California, Irvine

Here, we present light-induced dielectrophoresis as a label-free approach for characterizing heterogeneous cell lines, specifically human mesenchymal stem cells (hMSCs). This paper describes a protocol for using and optimizing a microfluidic device with a photoconductive layer to characterize the electrical behavior of hMSCs without altering their native state.

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Cancer Research

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research
Stephanie J. Hachey 1, Daniela Gaebler 1, Christopher C. W. Hughes 1,2
1Molecular Biology and Biochemistry, University of California, Irvine, 2Biomedical Engineering, University of California, Irvine

This protocol presents a physiologically relevant tumor-on-a-chip model to perform high-throughput basic and translational human cancer research, advancing drug screening, disease modeling, and personalized medicine approaches with a description of loading, maintenance, and evaluation procedures.

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Environment

Field Collection and Laboratory Maintenance of Canopy-Forming Giant Kelp to Facilitate Restoration
Phoebe D. Dawkins 1, Andrea Paz-Lacavex 2, Evan A. Fiorenza 1, Makena A. Rush 1, Rodrigo Beas-Luna 3, Julio Lorda 4, Luis Malpica-Cruz 5, Jose M. Sandoval-Gil 5, Tristin A. McHugh 6, Min K. Han 1, Matthew E. S. Bracken 1, Joleah B. Lamb 1
1Department of Ecology and Evolutionary Biology, University of California, Irvine, 2Department of Ecology and Evolutionary Biology, University of California, Santa Cruz, 3Facultad de Ciencias Marinas, Universidad Autónoma de Baja California, 4Facultad de Ciencias, Universidad Autónoma de Baja California, 5Instituto de Investigaciones Oceanológicas, Universidad Autónoma de Baja California, 6The Nature Conservancy

This protocol describes the field collection and regular laboratory maintenance of substrates seeded with canopy-forming giant kelp for use in restoration trials to address the success and limitations of the 'green gravel' technique in field settings.

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