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UT Southwestern Medical Center

15 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus
Xiaonan Dong 1, Pinghui Feng 2
1Center for Autophagy Research, Department of Internal Medicine, UT Southwestern Medical Center, 2Department of Microbiology, UT Southwestern Medical Center

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (γHV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for γHV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

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Medicine

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells
Ozlem Kulak 1, Lawrence Lum 1
1Department of Cell Biology, UT Southwestern Medical Center

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

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Immunology and Infection

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production
Junjie Zhang 1, Lining Zhu 1, Pinghui Feng 1
1Department of Microbiology and Immunology, Keck School of Medicine, University of Southern California

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (γHV68) that is closely-related to human Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. “Reconstituting” the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

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Biology

Rapid Analysis and Exploration of Fluorescence Microscopy Images
Benjamin Pavie *1, Satwik Rajaram *1, Austin Ouyang 2, Jason M. Altschuler 1,3, Robert J. Steininger III 1, Lani F. Wu 1, Steven J. Altschuler 1
1Green Center for Systems Biology, UT Southwestern Medical Center, 2Advanced Imaging Research Center, UT Southwestern Medical Center, 3Princeton University

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

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Neuroscience

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb
Wayne I. Doyle 1, Gary F. Hammen 2, Julian P. Meeks 1
1Department of Neuroscience, UT Southwestern Medical Center, 2Department of Anatomy and Neurobiology, Washington University in St. Louis

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

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Medicine

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling
Jerome D. Robin 1, Woody E. Wright 1, Yaqun Zou 2, Stacy A. Cossette 3, Michael W. Lawlor 3, Emanuela Gussoni 4
1Department of Cell Biology, UT Southwestern Medical Center, 2National Institute of Neurological Disorders and Stroke, National Institute of Health, 3Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, 4Division of Genetics and Genomics, Boston Children's Hospital

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

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Immunology and Infection

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies
S. M. Nashir Udden 1, Sumyya Waliullah 1, Melanie Harris 1, Hasan Zaki 1
1Department of Pathology, UT Southwestern Medical Center

The ex vivo organ culture allows investigation of biological processes in the context of the intact tissue architecture. Here, we introduce a method of ex vivo culture of the mouse colon, which can be used to study innate immunity and antimicrobial host defense in the intestine.

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Developmental Biology

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts
Antonio Fernandez-Perez 1, Nikhil V. Munshi 1
1Department of Internal Medicine- Cardiology, UT Southwestern Medical Center

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

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Biochemistry

Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers
Sarah Hanna-Addams 1, Zhigao Wang 1
1Department of Molecular Biology, UT Southwestern Medical Center

Here we use two-dimensional semi-denaturing agarose gel electrophoresis to confirm the presence of amyloid-like fibers of heterogeneous size and exclude the possibility that the size heterogeneity is due to dissociation of the amyloid fibers during the gel running process.

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Environment

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification
Owen Hudson 1, Sumyya Waliullah 1, Justin Hand 2, Romina Gazis-Seregina 3, Fulya Baysal-Gurel 4, Md Emran Ali 1
1Department of Plant Pathology, Coastal Plain Experiment Station, University of Georgia, 2University of Georgia Cooperative Extension, 3Department of Plant Pathology, Tropical Research & Education Center, University of Florida, 4Department of Agricultural and Environmental Sciences, Otis L. Floyd Nursery Research Center, Tennessee State University

We developed a method to detect Phytophthora capsici zoospores in water sources using a filter paper DNA extraction method coupled with a loop-mediated isothermal amplification (LAMP) assay that can be analyzed in the field or in the lab.

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Neuroscience

Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices
Katarina Stoklund Dittlau 1,2, Emily N. Krasnow 1,2, Laura Fumagalli 1,2, Tijs Vandoorne 1,2, Pieter Baatsen 3,4, Axelle Kerstens 3,4, Giorgia Giacomazzi 5, Benjamin Pavie 3,4, Elisabeth Rossaert 1,2, Jimmy Beckers 1,2, Maurilio Sampaolesi 5, Philip Van Damme 1,2,6, Ludo Van Den Bosch 1,2
1Department of Neurosciences, Experimental Neurology, and Leuven Brain Institute, KU Leuven - University of Leuven, 2VIB Bio Imaging Core, KU Leuven - University of Leuven, 3Department of Development and Regeneration, Stem Cell and Developmental Biology, KU Leuven - University of Leuven, 4Department of Neurology, University Hospitals Leuven

We describe a method to generate human motor units in commercially available microfluidic devices by co-culturing human induced pluripotent stem cell-derived motor neurons with human primary mesoangioblast-derived myotubes resulting in the formation of functionally active neuromuscular junctions.

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Medicine

Murine Orchiectomy and Ovariectomy to Reduce Sex Hormone Production
Ashley A. Rowe 1, Yacine Issioui 1, Blessing Johnny 1, Katherine J. Wert 1,2,3,4
1Department of Ophthalmology, UT Southwestern Medical Center, 2Department of Molecular Biology, UT Southwestern Medical Center, 3Hamon Center for Regenerative Science and Medicine, UT Southwestern Medical Center, 4Peter O'Donnell Jr. Brain Institute, UT Southwestern Medical Center

This manuscript describes a consistent way to quickly perform survival rodent orchiectomies and ovariectomies.

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Neuroscience

A Lightweight Drive Implant for Chronic Tetrode Recordings in Juvenile Mice
Robert J. Pendry 1,2, Lilyana D. Quigley 1,2, Lenora J. Volk 1,3,4, Brad E. Pfeiffer 1,3
1Department of Neuroscience, UT Southwestern Medical Center, 2Neuroscience Graduate Program, UT Southwestern Medical Center, 3O’Donnell Brain Institute, UT Southwestern Medical Center, 4Department of Psychiatry, UT Southwestern Medical Center

Here, we describe a micro-drive design, surgical implantation procedure, and post-surgery recovery strategy that allow for chronic field and single-unit recordings from multiple brain regions simultaneously in juvenile and adolescent mice across a critical developmental window from postnatal day 20 (p20) to postnatal day 60 (p60) and beyond.

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Bioengineering

4D Light-sheet Imaging of Zebrafish Cardiac Contraction
Xinyuan Zhang 1, Alireza Saberigarakani 1, Milad Almasian 1, Sohail Hassan 1, Manasa Nekkanti 1, Yichen Ding 1,2,3
1Department of Bioengineering, The University of Texas at Dallas, 2Center for Imaging and Surgical Innovation, The University of Texas at Dallas, 3Hamon Center for Regenerative Science and Medicine, UT Southwestern Medical Center

This protocol utilizes light-sheet imaging to investigate cardiac contractile function in zebrafish larvae and gain insights into cardiac mechanics through cell tracking and interactive analysis.

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Bioengineering

Light-Sheet Imaging to Reveal Cardiac Structure in Rodent Hearts
Milad Almasian 1, Alireza Saberigarakani 1, Xinyuan Zhang 1, Brian Lee 1, Yichen Ding 1,2,3
1Department of Bioengineering, The University of Texas at Dallas, 2Center for Imaging and Surgical Innovation, The University of Texas at Dallas, 3Hamon Center for Regenerative Science and Medicine, UT Southwestern Medical Center

The protocol utilizes advanced light-sheet microscopy along with adapted tissue clearing methods to investigate intricate cardiac structures in rodent hearts, holding great potential for the understanding of cardiac morphogenesis and remodeling.

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