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Tufts University

21 ARTICLES PUBLISHED IN JoVE

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Biology

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells
L Cristina Gavrilescu 1, Richard A Van Etten 1
1Molecular Oncology Research Institute, Tufts University

This technique demonstrates an efficient way to prepare replication-defective retroviral stocks encoding a human oncogene, and subsequently used for induction of myeloproliferative disease in the mouse model.

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Biology

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates
Lauren E. Frank 1, Angela D. Caldera-Siu 1, Emmanuel N. Pothos 1
1Department of Pharmacology and Experimental Therapeutics, Tufts University

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

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Biology

Survivable Stereotaxic Surgery in Rodents
Brenda M. Geiger 1, Lauren E. Frank 1, Angela D. Caldera-Siu 1, Emmanuel N. Pothos 1
1Department of Pharmacology and Experimental Therapeutics, Tufts University

The monitoring of extracellular neurotransmitter levels in distinct brain regions of freely moving animals offers insights on the link between neurotransmitter release and behavior. In vivo microdialysis coupled with electrochemical detection provides excellent anatomical and chemical resolution; and information on how basal neurotransmission is altered by pharmacological or physiological manipulations.

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Bioengineering

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering
Kathy Yuan Ye 1, Kelly Elizabeth Sullivan 1, Lauren Deems Black 1
1Department of Biomedical Engineering, Tufts University

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

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Bioengineering

Silk Film Culture System for in vitro Analysis and Biomaterial Design
Brian D. Lawrence 1, Zhi Pan 1, Michael D. Weber 1, David L. Kaplan 2, Mark I. Rosenblatt 1
1Margaret M. Dyson Vision Research Institute, Weill Cornell Medical College , 2Department of Biomedical Engineering, Tufts University

Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.

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Bioengineering

Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models
Duong D. Tu *1, Abhishek Seth *1, Eun Seok Gil 2, David L. Kaplan 2, Joshua R. Mauney 1, Carlos R. Estrada Jr. 1
1Children's Hospital Boston, Harvard Medical School, 2Tufts University

Surgical stages of bladder augmentation are described using 3-D scaffolds in murine and rat models. To test the efficacy of biomaterial configurations for use in bladder augmentation, techniques for both awake and anesthetized cystometry are presented.

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Neuroscience

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System
Haruki Higashimori 1, Yongjie Yang 1,2
1Department of Neuroscience, Tufts University, 2Neuroscience Program, Tufts Sackler School of Graduate Biomedical Sciences

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

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Chemistry

Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles
Shuai Nie 1, Elena Benito-Peña 1,2, Huaibin Zhang 1, Yue Wu 3, David R. Walt 1
1Department of Chemistry, Tufts University, 2Department of Analytical Chemistry, Complutense University (Spain), 3Department of Electrical and Computer Engineering, Tufts University

We describe a procedure for profiling salivary proteins using multiplexed microsphere-based antibody arrays. Monoclonal antibodies were covalently linked to fluorescent dye-encoded 4.5 μm polymer microspheres using carbodiimide chemistry. The modified microspheres were deposited in fiber-optic microwells to measure protein levels in saliva using fluorescence sandwich immunoassays.

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Behavior

Nest Building as an Indicator of Health and Welfare in Laboratory Mice
Brianna N. Gaskill 1, Alicia Z. Karas 2, Joseph P. Garner 3,4, Kathleen R. Pritchett-Corning 1
1Research Models and Services, Charles River, 2Department of Clinical Sciences, Tufts University, 3Department of Comparative Medicine, Stanford University, 4Department of Psychiatry and Behavioral Sciences, Stanford University

We demonstrate the utility of nest building behavior in laboratory mice as an indicator of welfare. Nest scoring is a sensitive technique that is altered by temperature, illness, and aggression. The time to integrate into nest test (TINT) is a simple cage-side assessment that can detect postoperative pain.

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Bioengineering

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue
Karolina Chwalek 1, Disha Sood 1, William L. Cantley 1, James D. White 1, Min Tang-Schomer 2, David L. Kaplan 1
1Department of Biomedical Engineering, Tufts University, 2Department of Pediatrics, University of Connecticut Health Center & Connecticut Children's Medical Center

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

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Neuroscience

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster
Timothy P. Murphy 1,2, Dan D. Luu 1, Jessica A. DeSimone 1,3, Thomas C. O'Brien 1,4, Christopher J. Lally 1,5, Jillian J. Lindblad 1,6, Sarah M. Webster 1
1Department of Biology, College of the Holy Cross, 2School of Medicine, Georgetown University, 3Department of Biochemistry, Giesel School of Medicine, Dartmouth College, 4School of Medicine, Tufts University, 5Transgenomic Inc., 6Department of Molecular, Cell and Cancer Biology, UMass Medical School

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

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Neuroscience

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons
Ji-Yong Kang 1, Daichi Kawaguchi 2, Lei Wang 3
1Department of Neuroscience, School of Medicine, Tufts University, 2Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 3Department of Pharmaceutical Chemistry and the Cardiovascular Research Institute, University of California, San Francisco

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

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Bioengineering

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays
Syrena C. Fernandes 1, Daniel J. Wilson 1, Charles R. Mace 1
1Department of Chemistry, Tufts University

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

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Immunology and Infection

A Method to Test the Efficacy of Handwashing for the Removal of Emerging Infectious Pathogens
Marlene K. Wolfe 1, Daniele S. Lantagne 1
1Department of Civil and Environmental Engineering, Tufts University

Handwashing is widely recommended to prevent infectious disease transmission. However, there is little evidence on which handwashing methods are most efficacious at removing infectious disease pathogens. We developed a method to assess the efficacy of handwashing methods at removing microorganisms.

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Immunology and Infection

Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers
Alyssa C. Fasciano 1, Sarah E. Blutt 2, Mary K. Estes 2, Joan Mecsas 3
1Program in Immunology, Sackler School of Graduate Biomedical Sciences, 2Department of Molecular Virology and Microbiology, Baylor College of Medicine, 3Department of Molecular Biology and Microbiology, Tufts University

This protocol describes how to induce the differentiation of M cells in human stem cell-derived ileal monolayers and methods to assess their development.

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Behavior

Using Practice Testing, Public Speaking, and Source Monitoring to Examine the Influences of Learning Strategies and Stress on Episodic Memory
Amy M. Smith 1, Elizabeth Race 2, F. Caroline Davis 1,3, Ayanna K. Thomas 2
1Center for Applied Brain and Cognitive Sciences, Tufts University, 2Department of Psychology, Tufts University, 3U.S. Army Combat Capabilities Development Command (CCDC) Soldier Center

The present experiment combined three experimental procedures — a retrieval-practice learning manipulation, a list-discrimination task, and a stress-induction technique — to examine the influences of different learning strategies and acute stress on multiple measures of episodic memory.

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Environment

A Gnotobiotic System for Studying Microbiome Assembly in the Phyllosphere and in Vegetable Fermentation
Esther R. Miller 1, Jonah O'Mara Schwartz 1, Grace Cox 1, Benjamin E. Wolfe 1
1Department of Biology, Tufts University

A method of growing germ-free Napa cabbages has been developed which enables researchers to evaluate how single microbial species or multispecies microbial communities interact on cabbage leaf surfaces. A sterile vegetable extract is also presented which can be used to measure shifts in community composition during vegetable fermentation.

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Cancer Research

In Vivo Targeting of Xenografted Human Cancer Cells with Functionalized Fluorescent Silica Nanoparticles in Zebrafish
Xiaodan Qin *1, Fabrice F. J. Laroche *1, Saquib Ahmed M. A. Peerzade 2, Andrew Lam 1, Igor Sokolov 2,3,4, Hui Feng 1
1Departments of Pharmacology and Medicine, Section of Hematology and Medical Oncology, The Cancer Research Center, Boston University School of Medicine, 2Department of Biomedical Engineering, Tufts University, 3Department of Mechanical Engineering, Tufts University, 4Department of Physics, Tufts University

Described here is a method for utilizing zebrafish embryos to study the ability of functionalized nanoparticles to target human cancer cells in vivo. This method allows for the evaluation and selection of optimal nanoparticles for future testing in large animals and in clinical trials.

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Biochemistry

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage
Candice M. Etson 1,2, Petar Todorov 1, Nooshin Shatery Nejad 2, Nirmala Shrestha 2, David R. Walt 1,3
1Department of Chemistry, Tufts University, 2Department of Physics, Wesleyan University, 3Wyss Institute at Harvard University

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

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Immunology and Infection

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness
Alexsandra Lenhard *1, Basma H. Joma *2,3, Nalat Siwapornchai 2, Anders P. Hakansson 4, John M. Leong 2,5, Elsa N. Bou Ghanem 1
1Department of Microbiology and Immunology, University at Buffalo School of Medicine, 2Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 3Graduate Program in Immunology, Tufts Graduate School of Biomedical Sciences, 4Department of Translational Medicine, Lund University, 5Stuart B. Levy Center for the Integrated Management of Antimicrobial Resistance, Tufts University

This paper describes a novel mouse model for the transition of pneumococcus from an asymptomatic colonizer to a disease-causing pathogen during viral infection. This model can be readily adapted to study polymicrobial and host-pathogen interactions during the different phases of disease progression and across various hosts.

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Genetics

Electrophoretic Analysis of Replication Through Structure-Prone DNA Repeats Within the SV40-Based Human Episome
Nicholas H. Mandel 1, Sergei M. Mirkin 1
1Department of Biology, Tufts University

Here, we outline the procedure for analyzing replication progression through pathogenic, structure-prone repeats using 2-dimensional gel electrophoresis.

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